General Microbiology Lab
General Microbiology Lab
General Microbiology Lab
MICROBIOLOGY
LABORATORY 2021
Alice Lee
North Carolina State University
North Carolina State University
MB352 General Microbiology Laboratory
2021 (Lee)
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1: LABORATORY SAFETY
1.1: SAFETY PROCEDURES FOR THE MICROBIOLOGY LABORATORY
1.2: BIOSAFETY LEVELS AND PPE
1.3: REVIEW QUESTIONS
REVIEW QUESTIONS II
REVIEW QUESTIONS III
2: CULTIVATION OF MICROBES
2.1: INTRODUCTION GROWTH MEDIA
2.2: INTRODUCTION TO BACTERIAL GROWTH AND ASEPTIC TECHNIQUES
2.3: EXAMPLES OF BACTERIAL GROWTH CHARACTERISTICS IN BROTHS, SLANTS AND PLATES
2.4: LAB PROCEDURES- PREPARE SOLID MEDIA, ASEPTIC TECHNIQUE, T-STREAKING
2.5: RESULTS
2.6: REVIEW QUESTIONS
3: MICROSCOPY
3.1: INTRODUCTION TO THE MICROSCOPE
3.2: COMPARISON OF SIZES AND SHAPES OF MICROORGANISMS
3.3: LAB PROCEDURES- OPERATING A MICROSCOPE
3.4: RESULTS
3.5: REVIEW QUESTIONS
4: STAINING TECHNIQUES
4.1: INTRODUCTION TO STAINING
4.2: SPECIALIZED BACTERIAL STAINING TECHNIQUES
4.3: LAB PROCEDURES- BACTERIAL SMEAR, SIMPLE AND GRAM STAINING
4.4: RESULTS
4.5: REVIEW QUESTIONS
5: ENUMERATION OF BACTERIA
5.1: INTRODUCTION TO ENUMERATION OF BACTERIA
5.2: LAB PROCEDURES- HOW TO OPERATE A PIPETTOR
5.3: LAB PROCEDURES- VIABLE PLATE COUNT
5.4: RESULTS
5.5: REVIEW QUESTIONS
6: MICROBIAL PHYSIOLOGY
6.1: INTRODUCTION TO OXYGEN REQUIREMENTS
6.1.1: DETERMINING OXYGEN REQUIREMENTS AND ANAEROBES
6.2: TEMPERATURE, PH, AND OSMOTIC REQUIREMENTS
6.3: BACTERIAL GROWTH DYNAMICS
6.4: BACTERIOPHAGES
6.5: LAB PROCEDURES- TESTING OXYGEN REQUIREMENTS
6.6: LAB PROCEDURES- PLAQUE ASSAY
6.7: RESULTS
6.8: REVIEW QUESTIONS
7: MICROBIAL METABOLISM
7.1: INTRODUCTION TO BIOCHEMICAL TESTS PART I
7.2: INTRODUCTION TO BIOCHEMICAL TESTS PART II
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7.3: LAB PROCEDURES- BIOCHEMICAL TESTS
7.4: RESULTS
7.5: REVIEW QUESTIONS
8: BACTERIAL IDENTIFICATION
8.1: INTRODUCTION TO BACTERIAL IDENTIFICATION USING CULTURE MEDIA
8.2: INTRODUCTION TO BACTERIAL IDENTIFICATION USING ENTEROTUBE TEST
8.3: INTRODUCTION TO BACTERIAL IDENTIFICATION USING GENOTYPIC METHODS
8.4: LAB PROCEDURES- ENTEROTUBE INOCULATION
8.5: LAB PROCEDURES- PCR AND GEL ELECTROPHORESIS
8.6: RESULTS
8.7: REVIEW QUESTIONS
BACK MATTER
INDEX
GLOSSARY
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CHAPTER OVERVIEW
1: LABORATORY SAFETY
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1.1: Safety Procedures for the Microbiology Laboratory
Learning Outcomes
Explain & practice safe microbiological procedures, protective measures, and emergency procedures.
Identify safe laboratory practices when working in a microbiology lab
Watch Video 1: start video at 9:55 - end video at 10:26, covers different types of bench surfaces and cleaning methods
NOTE: The following recommended practices and procedures for working safely on microbiology projects in a teaching
laboratory environment are based on “Guidelines for Biosafety in Teaching Laboratories,” from the American Society for
Microbiology (ASM). The full documents may be viewed at this URL: https://asm.org/getattachment/3c1eb3...Guidelines.pdf
BioSafety Levels
Handling microbes requires specialized laboratory facilities and techniques. Biosafety is the application of safety
precautions that reduce a scientists’ risk of exposure to a potentially infectious microbe and limit contamination of the
work environment. Bacteria pose varying degrees of risk both in a controlled laboratory environment and in their natural
settings. Therefore, the level of containment necessary for working safely with bacterial cultures also varies according to a
system that classifies microbes into one of four biosafety levels (BSL), which provides minimum standards for safe
handling of microbes at each level. BSLs are defined and containment practices are detailed by the Centers for Disease
Control and Prevention (CDC) for laboratories in the United States. The full document,
“BiosafetyinMicrobiologicalandBiomedicalLaboratories,” can be viewed in its entirety
at http://www.cdc.gov/biosafety/publications/bmbl5/index.htm.
We designate most of our labs under 4 special hazard categories called biosafety levels (BSLs). Each level has specific
controls for containment of microbes and biological agents. Each biosafety level builds on the controls of the level before
it. All of these levels follow “standard microbiological practices” : which are those practices common to all labs, which
include not eating, drinking, or applying cosmetics, washing hands after working in the lab, routinely decontaminating
work area.
BSL1: If you work in a lab that is designated a BSL-1, the microbes there are not known to consistently cause disease in
healthy adults and present minimal potential hazard to laboratorians and the environment. An example of a microbe that is
typically worked with at a BSL-1 is a nonpathogenic strain of E. coli. Work can be done on an open lab bench, requires a
sink. Requires personal protective equipment (PPE) such as lab coats, gloves, eye protection.
BSL 2: BSL-2 builds upon BSL-1. If you work in a lab that is designated a BSL-2, the microbes there pose moderate
hazards to laboratorians and the environment. The microbes are typically indigenous and associated with diseases of
varying severity. An example of a microbe that is typically worked with at a BSL-2 laboratory is Staphylococcus aureus. It
includes various bacteria and viruses that cause only mild disease to humans, or are difficult to contract via aerosols in a
lab setting, such as Clostridium difficile, most Chlamydiae, hepatitis A, B, and C, influenza A viruses, Salmonella. BSL-2
differs from BSL-1 in that: laboratory personnel have specific training in handling pathogenic agents and are directed by
scientists with advanced training; access to the laboratory is limited when work is being conducted; extreme precautions
are taken with contaminated sharp items; and certain procedures in which infectious aerosols or splashes may be created
BSL-4 labs builds on the containment requirements of BSL-3 and is the highest level of biological safety. BSL-4 labs is
required for work with dangerous and exotic agents that pose a high individual risk of life-threatening disease, aerosol
transmission or unknown risk of transmission. The microbes in BSL4 labs cause infections that are frequently fatal and
generally there are no vaccines or treatments for these infections. There are only a small number of such labs in the U.S
(<10) and the world. Laboratory staff must have specific and thorough training in handling extremely hazardous infectious
agents. Access to the laboratory is controlled by the laboratory supervisor. All handling of agents must be performed in a
gas tight Class III Biosafety Cabinet or by personnel wearing a positive pressure protective suit. BSL-4 Laboratories have
special engineering and design features to prevent microorganisms from being released into the environment. The lab is in
a separate building or isolated restricted zone of the building, and has a dedicated supply and exhaust air, as well as
vacuum lines and decontamination systems. Personnel mush change clothing before entering and shower upon exiting.
Most of the pathogens worked on are viruses: Crimean-Congo hemorrhagic fever caused by Ebola, Junin, Lassa, Machupo,
Marburg viruses , and tick-borne encephalitis virus complex (including Absettarov, Hanzalova, Hypr, Kumlinge, Kyasanur
Forest disease, Omsk hemorrhagic fever, and Russian Spring-Summer encephalitis).
Watch video 1
Video 1: proper usage of PPE. Your instructor will explain how to locate, use, and store your PPE in the lab.
Watch Video 1: WHO: Good Microbiological Practices & Procedures. Personal Protective Equipment (PPE) (19:41)
URL: https://youtu.be/Cuw8fqhwDZA
References:
1. WHO. World Health Organization Biosafety Series. https://www.who.int/ihr/publications...deo-series/en/
"I would literally fall asleep if I was having a conversation or doing anything that involved my brain," she says.
"I would literally fall asleep if I was having a conversation or doing anything that involved my brain," she says.
watch video 1: General lab safety by the Amoeba sisters. URL: https://youtu.be/MEIXRLcC6RA
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lab safety is important
Answer
Add texts here. Do not delete this text first.
Review this
Learning Objectives
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Learning Objectives
Explain the following laws within the Ideal Gas Law
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Example 1
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Review questions III
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CHAPTER OVERVIEW
2: CULTIVATION OF MICROBES
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2.1: Introduction Growth Media
Learning Outcomes
Recognize various types of growth media: solid, broth.
Understand the uses of selective and differential growth media
Determine the properties of some common bacterial types when grown on selective and differential growth media
Growth Media
To study bacteria and other microorganisms, it is necessary to grow them in controlled conditions in the laboratory. Growth
media contain a variety of nutrients necessary to sustain the growth of microorganisms. There are two commonly used
physical forms of growth media: liquid media and solid growth media. A liquid medium is called a broth (image 2).
Solid growth media usually contains agar (image 1), which is a mixture of polysaccharides derived from red algae. It is
used as a solidification agent because it (1) is not broken down by bacteria, (2) contains no nutrients that can be used by
bacteria and (3) melts at high temperatures, and yet is solid at temperatures used for most bacterial growth. Solid growth
media is used in the following forms: agar plates, agar slants, and agar deeps. To make agar deeps or agar slants, melted
agar is poured into a test tube and then allowed to solidify vertically (agar deep), or at a slant (agar slant). Agar plates are
made by pouring melted agar into a petri dish.
Image 1: Solid Agar slant Image 2: Broth media in test tube. Images by Anne Hanson, University of Maine,
Orono.
Figure 1:
Solid growth media forms
Broths can be used to determine growth patterns in a liquid medium, and for certain types of inoculations and metabolic
tests. They are also the method of choice for growing large quantities of bacteria. Agar slants are commonly used to
generate stocks of bacteria. Agar plates can be used to separate mixtures of bacteria and to observe colony characteristics
of different species of bacteria . Deeps are used for several different types of differential metabolic tests (e.g., the caseinase
hydrolysis test)
Example: A general purpose growth medium: e.g. tryptic soy agar (TSA) or Luria broth (LB) is used to grow a wide
variety of non-fastidious bacteria. This type of medium is often a complex growth medium.
Image 5: Staphylococcus aureus, a Gram positive organism, grows on this PEA plate while Serratia marcescens, a Gram
negative organism, does not. Image by WelcometoMicrobugz.
URL: https://www.austincc.edu/microbugz/p...cohol_agar.php
A differential growth medium is formulated such that different types of bacteria will grow with different
characteristics (e.g. colony color). An example of a differential growth medium is blood agar, which differentiates
among bacteria based on their ability to break down red blood cells and hemoglobin. Blood agar is also a complex
growth medium because it contains blood.
Image 6: Blood agar plate. Image by Rebecca Buxton, University of Utah, Salt Lake City, UT.
A growth medium can be both selective and differential. For example, EMB (eosin methylene blue agar) inhibits the
growth of Gram-positive bacteria. Gram negative bacteria that grow on this medium are differentiated based on their
ability to ferment the sugars lactose and sucrose. Strong fermenters of lactose or sucrose will produce large amounts of
acid and will appear dark purple to black. Escherichia coli, a strong fermenter will frequently appear as colonies with
Image 7: Eosin-methylene blue ( EMB) agar plate inoculated with Escherichia coli (a Gram-negative coliform bacterium)
showing good growth of dark blue-black colonies with metallic green sheen indicating vigorous fermentation of lactose
and acid production which precipitates the green metallic pigment. (Naowarat Cheeptham, Thompson Rivers University,
Kamloops, BC, Canada)
Making Media
I. Making culture media requires patience and attention to detail. Watch Video 1: Making Microbiological Media
II. Pouring Agar plates. Watch Video 2: Solid Media Preparation, video was filmed at NC State Microbiology labs.
Watch Video 1: Solid media preparation, video was filmed at NC State Microbiology labs.
URL:https://youtu.be/P7M_MCXbZjc
Differentiation
Yellow Halo Organism ferments mannitol Probable S. aureus
Staphylococcus species (other than S.
No Yellow Halo Organism does not ferment mannitol
aureus); Micrococcus (yellow colonies)
Eosin-methylene blue agar (EMB): Differential and selective growth medium. This medium contains peptone, lactose,
sucrose and the dyes eosin Y and methylene blue. Gram positive organisms are inhibited by the dyes, so this medium is
selective for Gram negative bacteria. The medium differentiates based on the ability to ferment lactose (and/or sucrose.)
Organisms that cannot ferment either of the sugars produce colorless colonies. Organisms that ferment the sugars with
some acid production produce pink or purple colonies; organisms that ferment the sugars and produce large amounts of
acid form colonies with a green metallic sheen. This medium is commonly used to detect the presence of fecal coliforms
(like E. coli)—bacteria that grow in the intestines of warm-blooded animals. Fecal coliforms produce large amounts of
acid when fermenting lactose and/or sucrose; non-fecal coliforms will produce less acid and appear as pink or purple
colonies.
Table 2: EMB Agar
Result Identification Interpretation
References
1) Dr. Gary Kaiser (COMMUNITY COLLEGE OF BALTIMORE COUNTY, CATONSVILLE CAMPUS)
Image 1: Notice individually isolated colonies: Large, creamy white, circular, beta-hemolytic colonies typical
of Staphylococcus aureus cultured on Blood agar. Image by Rebecca Buxton, University of Utah, Salt Lake City, UT.
Image 2: Example of circular formed colonies--Serratia marcescens colonies cultivated on trypticase soy agar. Image by
Bryan MacDonald, Christopher Adams, and Kyle Smith, Brigham Young University, Provo, UT.
Image 3: Undulate form colonies-- Streak plate isolation of Mycobacterium smegmatis on trypticase soy agar (TSA)
incubated for 96 hours at 37oC. Note the rough texture of colonies characteristic of this organism. Image by Tasha L.
Sturm, Cabrillo College, Aptos, CA.
Watch Video 1: Aseptic Techniques tips. Video by Dr. Gary Kaiser (CCBC). (5:56) URL: https://youtu.be/_tMM0F0Pr60
Image 5: Agar plate with T-streak of E. coli . This is a pure culture and notice the isolated colonies in the last sector of the
plate, they appear as single, individual, round colonies. Image by Rebecca Buxton, University of Utah, Salt Lake City, UT.
Watch video 3: Isolating bacterial colonies using a T-streak. Excellent summary video of many things we talked about in
this section: the difference between a cell vs a colony, how to separate bacterial species using a T-streak isolation method,
common mistakes when doing a T-streak, and the results of a T-streak. They use disposable loops here and sterilize the
loop with 70% ethanol between each section when doing the T-streak. You can also discard the loop (in biohazard) when
streaking between sections, or use a metal loop (usually made of nickel-chrome wire) that you sterilize using a bunsen
burner or a bacticinerator. Video by ID laboratory videos (9:17) URL: https://youtu.be/c1onYow0O58
Lab 1: T-Streak
Watch video 4: How to perform a T-streak for isolated colonies. This video was filmed in the Microbiology laboratories at
NC State. It will familiarize you with the use of a bacticinerator for sterilizing a metal inoculating loop. (7:39)
URL:https://youtu.be/NsQv7QOmdXo
Aseptic Technique
The average human is made up of approximately 1013 eukaryotic animal cells and 1014associated eukaryotic and
prokaryotic microbial cells. This means that 90% of "your" cells are not human.
The purpose of this exercise is to familiarize you with aseptic techniques. Aseptic technique can be thought of as the
manipulation of sterile instruments or culture media in such a way as to maintain sterility.
In nature, microorganisms almost always exist as mixed populations of many widely differing types. However, before most
properties and characteristics of a particular organism can be determined, the organism must first be isolated in pure
culture. A pure culture is one that contains a single strain of microorganism. For example, a pure culture of the bacterium
Escherichia coli contains only Escherichia coli cells of a particular strain - no other living microorganisms are present.
The term strain refers to a population of cells all descended from a single cell. The isolation and maintenance of pure
cultures is one of the most important procedures in microbiology, and one with which all students must be familiar.
Like all other organisms, microorganisms require nutrients and a favorable environment to grow and multiply. Microbes
are generally grown in a culture medium that contains essential nutrients and provides a suitable environment (e.g. proper
pH). Microbes are essentially grown in a liquid medium, broth, or on a solid medium, agar.
Since most laboratory studies are made with pure cultures, it is necessary to sterilize culture media, that is, completely
eliminate all living organisms. This medium must be maintained in a sterile condition, free from living organisms, until
inoculated with a pure culture.
To grow a microbial culture in a sterilized medium, a number of the cells, the inoculum, are transferred, inoculated, into or
onto the medium with special precautions to maintain the purity of the culture. The procedures used in the microbiology
laboratory to prevent contamination of pure cultures are commonly referred to as aseptic technique.
The general rules in following aseptic technique are: 1) put nothing into sterile material that is not itself sterile, except the
specific organism you are studying and, 2) do not expose sterile materials to sources of contamination, for example,
laboratory air.
Following inoculation, a microbial culture is grown (incubated) in a specific environment favorable for growth. Growth of
microbes is usually defined in terms of population growth, an increase in numbers of cells in the population, as opposed
to cellular growth, an increase in the size of an individual cell. The resultant growth is visible as cloudiness, turbidity, in a
liquid medium or as an isolated mass, colony, on a solid medium.
Materials:
Cultures
Escherichia coli (broth culture)
Serratia marcescens
mixed broth culture of Escherichia coli and Serratia marcescens
Media
one bottle sterile Nutrient agar
three tubes sterile Nutrient broth
The purpose of this exercise is to familiarize you with aseptic techniques. All the procedures have been designed to
minimize contamination. Observe the demonstration by your laboratory instructor and follow the procedures outlined in
the manual exactly. Place the bacticinerator in front of you and put all tubes and other equipment in a suitable location that
will allow you to reach them without any difficulty and without burning yourself.
At first these procedures for manipulating the loop, tubes, and caps will be difficult, but with practice these
manipulations will become more rapid and less cumbersome.
Operation of the Bacticinerator:
The bacticinerator is a gasless, flameless, heat sterilizer for use with inoculating loops, needles, glass tube/pipette mouths
and various metal & borosilicate glass instruments. Sterilization is accomplished through infrared heat. OPERATION:
Turn on the toggle switch to the “High” position; the red indicator light will come on when the switch is in the correct
position. Sterilizer must be heated for 15mins prior to use. Holding the inoculating loop by the handle, gently insert
inoculating loop to be sterilized into cylindrical sterilization area (see image below). Always ensure that the item is held
with an insulated material. Do not directly touch the sterilized item. Do not “park” the loop in there unintended, it will
MELT. Avoid scraping sides of element to ensure the longevity of the loop and heater element. Hold loop for 5
seconds. It is not necessary for loop to be glowing. The outer, metal shield of the heater element can reach temperatures
as high as 400°F. Do not touch outer metal shield or introduce any flammable or volatile materials in the area
surrounding the sterilizer.
Turn off bacticinerator after you have finished using it for the lab exercises for the day.
Lab Procedures:
A. Preparation of Solid Media
Solid medium is usually made by adding a solidifying agent to a broth medium. The most common solidifying agent is
agar, a substance obtained from marine algae and available in dried purified form. Although different agars vary
considerably in their physical properties, the usual melting point is 97-100°C. Thus, solid agar can be added to liquid
culture media and melted during heat sterilization. After sterilization, this liquid media can be poured into test tubes,
bottles, or petri dishes. On cooling, this medium containing agar solidifies at about 42°C. Once solidified, agar media may
be incubated over the entire range of temperatures a bacteriologist is likely to use (up to 70°C, perhaps) without melting.
An agar slant results when the hot molten agar media is allowed to harden in a test tube held in a tilted position. Pouring a
molten agar medium into plastic or glass Petri dishes makes agar plates.
5. While still holding the cap, grasp the bottle securely with the thumb and forefinger of your right hand. See Figure 1.
Lightly pass the lip of the bottle in front of the bacticinerator to burn off any adhering dust and to also to set up a negative
air current. The lip of the bottle would be sterile from the autoclaving procedure.
6. Lift the lid of a petri plate with your left or right hand just enough to allow you to pour some of the agar medium into
the bottom of the petri plate. See Figure 1-1b.
7. Pour enough medium into the petri plate bottom to just cover the bottom surface. (You should be able to easily pour four
plates and possibly five with 100 mL of medium.)
8. Replace the lid to cover the bottom of the petri plate.
9. Carefully and slowly slide the poured plates to an undisturbed (back) portion of your bench-top to cool and solidify. Do
not disturb these plates for at least 20 minutes.
10. These plates will be used in part D of this exercise.
B. Aseptic Transfers
To the microbiologist, transfer of cultures from tube to tube and from agar plates to tubes is a common and simple
procedure but requires careful attention to certain details. Transfer of microorganisms is often done using a wire loop.
Proper use of this inoculating loop will help insure maintenance of a pure culture.
1. Label the 3 tubes of sterile Nutrient broth. Label one "sterile control", one "broth transfer", and one "colony transfer".
2. The broth cultures of Escherichia coli and plate culture of Serratia marcescens are at the end of
your bench.
4. Now insert the loop into the E. coli broth culture and then remove it, carrying out a loopful of culture from the tube. See
Figure 2. Replace the tube cap and return the tube to the storage rack.
5. Pick up and remove the cap from the tube of sterile Nutrient broth labeled "broth transfer" in the same manner as
outlined in step 3a above.
6. Tilt the tube at about a 45°angle and insert the loop containing the culture into the open tube. Immerse the loop in the
broth, swirl the loop gently, then remove it. Pass the tube in front of the bacticinerator and replace the cap and return the
tube to the storage rack.
7. Sterilize the loop before putting it down by repeating step 3. This is necessary to avoid contaminating of the bench with
the culture.
8. Now, with your other hand, lift the lid of the petri plate containing a pure culture of the Serratia marcescens. Obtain an
inoculum by JUST TOUCHING ONE of the isolated single colonies on the agar surface. Do not dig into the agar. Do
not scrape the loop across the surface of the plate. Replace the lid of the plate immediately.
9. Pick up and remove the cap from the tube of sterile Nutrient broth labeled "colony transfer" in the same manner as
outlined in step 3a above.
10. Repeat steps 6 and 7.
11. To test your ability to properly sterilize your loop, pick a loopful of the culture from the E. coli broth culture tube
following the procedures outlined in steps 3, 3a, 4. Properly sterilize the loop containing the bacterial culture, as in step 7.
12. Pick up and remove the cap from the tube of sterile Nutrient broth labeled "sterile control" in the same manner as
outlined in step 3a above and insert the loop into the broth as described in step 6.
13. Repeat steps 11 and 12 a few times with the same "sterile control" tube.
C. Streak Plates
The streak plate technique is a rapid and simple method of isolating bacteria by mechanically spreading them over the agar
surface of a petri plate. The purpose is to obtain well-isolated colonies, each arising from a single bacterium, so that a
2. Label the bottom of each of the 3 prepared Nutrient agar plate with your name or initials, your lab section, and the
bacterial species to be used. In addition, label one "from broth", one "from agar", and one "mixed
culture".
3. Use the aseptic techniques you learned in part B. Sterilize the loop and allow it to cool.
Obtain an inoculum of bacterial cells from:
• the broth tube containing a pure culture of Escherichia coli
• the agar plate of Serratia marcescens
b) Beginning at the edge of the plate near the initial inoculum deposit, make
several (10-20) parallel streaks passing through the original inoculum
deposit. Cover one-third of the plate, starting at the edge and moving
towards the center of the plate. Do this by swinging the loop holder back
and forth on the surface of the agar with very gentle and even pressure,
using only a wrist motion. The loop should almost retrace its path with
each swing as it moves down the agar surface from the edge towards the
center of the plate. Try to keep the loop, wire, and loop holder in a plane
parallel to the surface of the agar throughout the streak. Try not to lift the
loop off of the agar. Try to keep the loop in contact with the surface from
the beginning of the streak to the end. Do not dig the loop into the agar
medium,
* Sterilize the loop and allow it to cool as described above.
c) Rotate the plate 90°.
Pull the loop through one edge of the previous streaks 2 or 3 times to re-inoculate the loop. Now streak the second third of
the plate with several (10-20) more parallel streaks again starting at an edge and moving towards the center of the plate.
After the initial re-inoculation, avoid touching the already streaked first third of the plate.
*Sterilize the loop and allow it to cool as described above.
d) Rotate the plate 90°.
Pull the loop through one edge of the streaks in the second third of the plate to re-inoculate the loop. Now streak the last
third of the plate as above, being careful to avoid the already streaked first and second thirds of the plate.
*Sterilize the loop after the last streaking.
D. Ubiquity of Microorganisms
Microorganisms are everywhere. The laboratory is populated with many different types of microbes, in the air, on the
benches, on the floor, and on your clothing and body. This omnipresence of microorganisms is one reason why you have
learned good aseptic techniques in parts A, B, and C of this exercise. This part of the exercise is designed to give you some
idea of the kind and numbers of microbes that are present in the laboratory. There is also some freedom to allow you to
satisfy your curiosity about the microorganisms all around you. Finally, you will be required to isolate one of the bacterial
species you obtain from this exercise for use in the next two exercises.
1. Check to be sure the agar has solidified in your plates from part A by carefully tilting the plates and looking for
movement. If the agar is solid, you may work with the plates. Label one plate on the bottom as "sterile control". Do not
open this plate at any time. This is used to check if you have good aseptic technique when you poured your plates.
2. Use one plate to test for the natural contamination of articles in your environment. Label this your “test plate” and with
the source “finger, cell phone, etc.” Suggested experiments include: lightly rubbing your fingers (no gloves on) over the
agar surface of the plate, call this the “test plate, finger”, or opening a plate to the air for 15-20 minutes, or coughing into
a plate, or gently wiping the agar surface with an object from your pocket or backpack. It is also possible to sample a
surface, such as the bench-top, floor, sink, clothing, or skin. If you wish to do so, wipe the surface with sterile cotton swab
then streak the entire agar surface of the plate with the swab. Use a light touch. The swabs are packaged individually and
are sterile until you contaminate them with something of your choosing. Make sure the cotton tip of the swab does not
come in contact with anything other than the surface you are testing. A moist swab works best for collecting samples from
a dry surface. You can moisten a swab on the surface of the nutrient agar plate (at a corner), then sample the area of
interest.
E. Incubation
In order for microorganisms to grow they need a suitable environment. One of the most critical environmental parameters
affecting growth of microorganisms is temperature. This will be discussed in lecture and is part of a later exercise. For
most organisms used in this laboratory,
30°C is an acceptable temperature for growth. Constant growth temperatures are maintained by incubating microbial
cultures in thermostatically controlled rooms or small ovens called incubators.
1. Incubate all tubes from part B in the 30°C incubator. Make sure labeling of your tubes is clear and complete. Place
tubes in racks provided by your instructor.
2. Incubate all plates from parts C and D in the 30°C incubator. Make sure labeling of your plates
is clear and complete.
Because of the high concentration of water in the agar medium, some water condensation forms in petri plates during
incubation. If plates were incubated right side up, moisture would likely drip from the cover to the surface of the agar and
spread out. This would disrupt individual colony formation and result in a confluent mass of growth. To avoid this, petri
plates are routinely inverted (bottom-side on top) during incubation.
F. Observation
Detailed observations are the key to performing, understanding, and designing scientific experiments. A keen eye and an
attention to subtle differences will help you successfully complete the laboratory report sheets. After incubation, plates and
tubes should be observed for evidence of microbial growth. Record your observations on the outlined report sheets
provided. These are only a guide, feel free to modify or add to them, as you deem necessary. Remember these report sheets
constitute your lab notebook and are the basis for lab grades and evaluations. They should be kept up to date and brought
to lab at all times.
DO NOT DISCARD YOUR SAMPLES UNTIL YOU ARE SURE OF THE RESULTS AND ARE CERTAIN THE
SAMPLES ARE NOT NEEDED FOR THE NEXT EXERCISE.
Watch Video 1: Aseptic techniques Inoculating broth, slant, and stab tubes
Watch Video 1: Aseptic techniques Inoculating broth, slant, and stab tubes. URL: https://youtu.be/nMoM8Ku5-8A
Lab 1: T-Streak
Watch Video 2:How to perform a T-streak for isolated colonies. This video was filmed in the Microbiology laboratories at
NC State. URL:https://youtu.be/NsQv7QOmdXo
A. Pouring plates:
Observe that your plates were poured properly--the bottom of the petri dish should be about half full and the media should
be sterile, even, and contain few or no bubbles.
B. Aseptic transfers
Look for turbidity in the broth indicating growth of microorganisms. Record the turbidity of the Nutrient broth in the three
tubes as + or -. Recall what your sterile Nutrient broth looked like before it was inoculated.
"sterile control"
"broth transfer"
"colony transfer"
Also note any other distinguishing characteristics (amount and location of turbidity, color, etc.) of the bacterial growth in
the broth and note and record any differences between species of bacteria. An effective method for recording your results
is to draw a picture of the tube and the appearance of the broth due to any growth.
From Broth: Describe growth characteristics and make a sketch in the circle below.
From Agar: Describe growth characteristics and make a sketch in the circle below.
2. Were you able to separate and identify the two different bacterial species in your "mixed culture"?
3. Colony morphology can be an aid in the identification of microorganisms.
Although colony morphology cannot be employed as the sole identifying criterion, it is a useful trait in the classification of
many common types of microorganisms. Five parameters are normally used to describe microbial colonies growing on an
agar surface.
a. Size: pinpoint, small, medium, or large; range: < l mm - 3cm
b. Color: absolutely white, various degrees of pigmentation
c. Texture: the texture of the colony as determined by touching the colony
with a needle; smooth (buttery), dry (granular), or mucoid (slimy) and the
appearance as judged by the manner in which the colony refracts light;
clear, glistening, dense, opaque, or translucent.
Compare your bacterial colonies to those of your bench mates. Note and record any differences in the way various
bacterial species grow on agar surfaces.
Escherichia coli:
Serratia marcescens:
D. Ubiquity of Microorganisms
Do not throw these test plates away until you have read Exercise 2, part D ("test plate isolate”).
Check to see if your "sterile control" plates remained sterile and were not contaminated. Observe, record, and DESCRIBE
the numbers and varieties of different microbial colonies that appear on your test plate. Indicate the sample source (finger
plate or other) or exposure method for the test plate.
"sterile control":
2. Why is it necessary to sterilize the loop between streaks when streaking for single colonies?
5. What medium would you use (TSA, EMB, MSA) if you wanted to determine if a Staphylococcus isolate could ferment
mannitol? Describe what you would see on this medium.
6. If you were testing water for the presence of fecal coliforms, what sort of medium would you use: TSA, EMB agar or
MSA agar? If fecal coliforms were present, what would their growth characteristics be on this medium?
1 2/12/2022
3.1: Introduction to the Microscope
Learning Outcomes
Review the principles of light microscopy and identify the major parts of the microscope.
Learn how to use the microscope to view slides of several different cell types, including the use of the oil immersion
lens to view bacterial cells.
Early Microscopy
The first microscope was developed in 1590 by Dutch lens grinders Hans and Zacharias Jansen. In 1667, Robert Hooke
described the microscopic appearance of cork and used the term cell to describe the compartments he observed. Anton van
Leeuwenhoek was the first person to observe living cells under the microscope in 1675—he described many types of cells,
including bacteria. Since then more sophisticated and powerful scopes have been developed that allow for higher
magnification and clearer images.
Microscopy is used by scientists and health care professionals for many purposes, including diagnosis of infectious diseases,
identification of microorganisms (microscopic organisms) in environmental samples (including food and water), and
determination of the effect of pathogenic (disease-causing) microbes on human cells. This exercise will familiarize you with
the microscopes we will be using to look at various types of microorganisms throughout the semester.
The basic unit of measurement of length in the metric system is the meter.
There are 1000 micrometers (microns, or µm) in one millimeter. 1 µm = 10-6 meter.
The microscope is one of the microbiologist's greatest tools. It allows for the visualization of small particles, including
microbes, which individually are too small to be seen with the human eye. With the help of proper illumination, a microscope
can magnify a specimen and optically resolve fine detail. This introduction to microscopy will include an explanation of
features and adjustments of a compound brightfield light microscope, which magnifies images using a two lens system.
Before reading the following discussion of the theory of the microscope, please familiarize yourself with the names of the
microscope parts shown in Figure 2 and their function.
1. Eyepiece/Ocular lens: Lens in which the final magnification occurs. Often is at 10X magnification, but can be different.
2. Revolving nose piece: Holds multiple objective lenses in place. The base of the nose piece can rotate, allowing each of the
lens to be rotated into alignment with the ocular lens.
3. Objective lenses: Initial magnification of your specimen occurs here. Most brightfield light microscopes have 3 objective
lenses seated into the resolving nose piece base.
The Optical System. The optical system of a compound microscope consists of two lens systems: one found in the
objective(s) lens(es) (Fig. 2, part 3); the other in the ocular (eyepiece) (Fig. 2 part 1). The objective lens system is found
attached to a rotating nosepiece (Fig. 2, part 2). A microscope usually has three or four objectives that differ in their
magnification and resolving power. Magnification is the apparent increase in size of an object. Resolving power is the term
used to indicate the ability to distinguish two objects as separate. The most familiar example of resolving power is that of car
headlights at night: at a long distance away, the headlights appear as one light; as the car approaches, the light becomes
For example, with a 10X objective lens and a 10X ocular, the total magnification of the microscope is 100X. If the objective
lens is changed to a 20X objective, then the total magnification is now 200X, whereas if a 10X objective is used with a 12.5X
ocular lens, the total magnification is now 125X. The use of objective and ocular lenses with different magnifications allows
greater flexibility when using the compound microscope. Due to the size of most bacteria (ranges widely from ~1um to over
100um), generally we require the use of the 100x oil immersion lens with a 10x ocular lense to view bacteria in a standard
brightfield light microscope.
The Illumination System. The objective and ocular lens systems can only perform well under optimal illumination
conditions. To achieve these conditions, the light from the light source (bulb) must be centered on the specimen. (In most
inexpensive microscopes, the manufacturer adjusts this centering. In more versatile microscopes, the centering becomes more
critical and is a function performed by the operator.) The parallel light rays from the light source are focused on the specimen
by the condenser lens system (see Fig. 2) The condenser can move up and down to affect this focus. Finally, the amount of
light entering the condenser lens system is adjusted using the condenser diaphragm. It is critical that the amount of light be
appropriate for the size of the objective lens receiving the light. This is important to give sufficient light, while minimizing
glare from stray light, which could otherwise reduce image detail. The higher the magnification and resolving power of the
lens, the more light is needed to view the specimen.
Objective lenses used for observing very small objects such as bacteria are almost always oil immersion lenses. With an oil
immersion lens, a drop of oil is placed between the specimen and the objective lens so that the image light passes through the
oil. Without the oil, light passing through the glass microscope slide and specimen would be refracted (bent) when it entered
the air between the slide and the objective lens. This refracted light might still be able to contribute to the image of the
specimen if the objective lens is large. However, at the higher magnification, the objective lens is small, so is unable to capture
this light. The loss of this light leads to loss of image detail. Therefore, at higher magnifications, the area between the slide and
the lens is modified to have the same (or nearly the same) refracting qualities (refractive index) as the glass and specimen by
the addition of immersion oil. Watch this NC BioNetwork video (https://youtu.be/-0EvnroWpVc) on oil immersion. For more
information, read this article (https://www.microscopeworld.com/t-us...rsion_oil.aspx).
To use an oil immersion lens, place a drop of oil on top of the dried specimen on the slide and carefully focus the microscope
so that the objective lens is immersed in the oil. Any lens, which requires oil, is marked "oil" or "oil immersion." Conversely,
any lens not marked "oil" should NOT be used with oil and is generally not sealed against oil seeping into and ruining the
objective.
Key Terms
microorganism, magnification, resolution, working distance, parfocal, parcentric, prokaryotic, eukaryotic, bacillus, coccus,
spirillum, spirochete, morphology, bacterial arrangements, depth of field, field of view, taxonomic classification
References:
Contributed by Joan Petersen & Susan McLaughli: Associate Professors (Biological Sciences and
Geology) at Queensborough Community College
Lumen Learning: Figure 3: Brightfield light microscope https://courses.lumenlearning.com/mi...of-microscopy/
Image 1: These are the different shapes of bacteria and their sizes compared with the width of a human hair.
The unit “μm” is a measurement of length, the “micrometer,” or commonly known as the micron. It
equals 1×10−6 meters that is, one millionth of a meter. Image courtesy of Kestin Schulz, Mariya W. Smit, Lydie Herfort
and Holly M. Simon URL: https://commons.wikimedia.org/wiki/F...omparisons.jpg
The cocci come in 5 different arrangements; the bacilli in 3 different arrangements; and the spirals in 3 different
forms.
1. Coccus
c. coccobacillus: oval and similar to a coccus (see Fig 2A, 2D, and 2E)
A single bacillus is typically 0.5-1.0 µm wide and from 1- 4 µm long. Small bacilli or bacilli that are
dividing or have just divided by binary fission may at first glance be confused for diplococci or
cocci (see Fig. 2A) so they must be observed carefully. You will, however, be able to see bacilli that have
not divided and are definitely rod-shaped as well as bacilli in the process of dividing.
3. Spiral
Spiral-shaped bacteria occur in one of three forms (see Fig. 3A):
a. vibrio: an incomplete spiral or comma-shaped (see Fig. 3A and Fig. 3B)
- Photomicrograph of a vibrio
- Scanning electron micrograph of Vibrio cholerae; courtesy of Dennis Kunkel's Microscopy
b. spirillum: a thick, rigid spiral (see Fig. 3A and Fig. 3C)
- Photomicrograph of a spirillum
c. spirochete: a thin, flexible spiral (see Fig. 3A and Fig. 3D)
- Photomicrograph of a spirochete
- Scanning electron micrograph of the spirochete Leptospira; courtesy of CDC
- scanning electron micrograph of the spirochete Treponema pallidum; courtesy of CDC
The spirals you will observe range from 5-40 µm long but some are over 100 µm in length. The spirochetes
are the thinnest of the bacteria, often having a width of only 0.25-0.5 µm.
To view a nice interactive illustration comparing size of cells and microbes, see the Cell Size and Scale
Resource at the University of Utah.
B. YEASTS
Yeasts, such as the common baker's yeast Saccharomyces cerevisiae (see Fig. 4), are unicellular fungi. They
usually appear spherical and have a diameter of 3 - 5 µm. Yeasts commonly reproduce asexually by a process
called budding. Unlike bacteria, which are prokaryotic, yeasts are eukaryotic.
- Scanning electron micrograph of Saccharomyces; courtesy of Dennis Kunkel's Microscopy
To view a nice interactive illustration comparing size of cells and microbes, see the Cell Size and Scale
Resource at the University of Utah.
C. MEASUREMENT OF MICROORGANISMS
The approximate size of a microorganism can be determined using an ocular micrometer (see Fig. 5) , an
eyepiece that contains a scale that will appear superimposed upon the focused specimen.
To view a nice interactive illustration comparing size of cells and microbes, see the Cell size & scale resource at
the University of Utah's Learn.genetics site, URL: https://learn.genetics.utah.edu/content/cells/scale/
Bacillus megaterium
Micrococcus luteus
E. Demonstration slide
Questions:
1.What are the structures seen on the end of these cells?
2. What is their function?
1 2/12/2022
4.1: Introduction to Staining
Learning Objectives
Describe the differences between simple staining and differential staining techniques.
Discuss how to prepare a bacterial smear from cultured organisms.
Distinguish between Gram-positive and Gram-negative bacteria.
Describe the process of the Gram stain procedure.
Use microscopy to examine Gram stained cells.
Describe select special staining procedures and view examples of these under oil immersion.
Image 1: Microscopic view of Bacillus (rod) shaped bacteria simple stained with crystal violet. Isolated and imaged by
Muntasir Alam, University of Dhaka, Department of Microbiology in
2007. https://commons.wikimedia.org/wiki/F...micrograph.jpg
Watch Video 1: How to apply a simple stain to a bacterial culture by NC BioNetwork (4:05)
URL: https://youtu.be/8ODeT9DLHKI
Image 2: Microscopic image of a Gram stain of mixed Gram-positive cocci (Staphylococcus aureus ATCC 25923, purple)
and Gram-negative bacilli (Escherichia coli ATCC 11775, red). Magnification:1,000. Image by Y
Tambe. https://commons.wikimedia.org/wiki/F...m_stain_01.jpg
Watch Video 2: Gram Stain Animation and discussion on what is happening at each step.
Watch Video 2: Gram stain animation with description of each step and interpretation of what is happening in each step by
Dr.G Bhanu Prakash Animated Medical Videos. (3:37) URL https://youtu.be/AZS2wb7pMo4
Watch Video 2: Gram Staining procedure, filmed at NC State Microbiology Labs. (5:58). URL: https://youtu.be/H-
fxk1be1hQ
Special Stains
There are a variety of staining procedures used to identify specific external or internal structures that are not found in all
bacterial species, such as a capsule stain and a flagella stain. For images and more examples of specialized stains, see
below and in the next section, 4.2 specialized bacterial staining techniques.
Capsule Stain
Some bacteria secrete a polysaccharide-rich structure external to the cell wall called a glycocalyx. If the glycocalyx is thin
and loosely attached, it is called a slime layer; if it is thick and tightly bound to the cell, it is called a capsule. The
glycocalyx can protect the cell from desiccation and can allow the cell to stick to surfaces like tissues in the body. They
may also provide cells with protection against detection and phagocytosis by immune cells and contribute to the formation
of a biofilm: in this way a glycocalyx can act as a virulence factor; (contributes to the ability of an organism to cause
disease).
Capsules can be detected using a negative staining procedure in which the background (the slide) and the bacteria are
stained, but the capsule is not stained. The capsule appears as a clear unstained zone around the bacterial cell. Since
capsules are destroyed by heat, the capsule staining procedure is done without heat-fixing the bacteria.
Silver Stain
Flagella (long whip-like structures used for bacterial motility) and some bacteria (e.g. spirochetes) are too thin to be
observed with regular staining procedures. In these cases, a silver stain is used. Silver nitrate is applied to the bacteria
along with a special mordant; the silver nitrate precipitates around the flagella or the thin bacteria, thus thickening them so
they can be observed under the light microscope.
Simple Stains:
Commonly used dyes for simple stains: Crystal Violet, Methylene Blue, Safranin
Uses one dye
Used to provide color to otherwise transparent bacterial cells
Can be used to determine cell size, morphology and arrangement
All bacteria are the same color when stained with the single dye that is used
Image 1: Simple stain with crystal violet showing rod shaped bacteria. Note that all bacteria are the same color of the dye,
crystal violet (purple), regardless of its cell wall composition. Image by Muntasir du, 2007
(https://commons.wikimedia.org/wiki/F...micrograph.jpg)
Gram Stain
Primary stain – crystal violet
Mordant – iodine; decolorizer- 95% Ethanol
Counterstain – Safranin
Common differential stain
Gram reaction (positive or negative) reflects cell wall properties
Also used to determine cell size, morphology and arrangement
Image 2. Gram positive, rod, Bacillus subtilis. (Ann C. Smith, University of Maryland, College Park, MD)
Acid-Fast Stain
The acid-fast stain is a differential stain used to identify acid-fast organisms such as members of the
genus Mycobacterium .
Acid-fast organisms are characterized by wax-like, nearly impermeable cell walls; they contain mycolic acid and large
amounts of fatty acids, waxes, and complex lipids. Acid-fast organisms are highly resistant to disinfectants and dry
conditions. (1)
Because the cell wall is so resistant to most compounds, acid-fast organisms require a special staining technique. The
primary stain used in acid-fast staining, carbolfuchsin, is lipid-soluble and contains phenol, which helps the stain penetrate
the cell wall. This is further assisted by the addition of heat. The smear is then rinsed with a very strong decolorizer,
which strips the stain from all non-acid-fast cells but does not permeate the cell wall of acid-fast organisms. The
decolorized non-acid-fast cells then take up the counterstain. (1)
Primary stain – Carbol fuchsin
Decolorizer – acid alcohol
Counterstain – Methylene blue
A differential stain used to detect bacteria with mycolic acid cell walls (genera Mycobacterium and Nocardia)
Developed to detect the bacterial species that causes tuberculosis
Acid-fast organisms resist decolorization with acid-alcohol
Image 4. A mixed culture of Mycobacterium smegmatus (acid-fast, red/pink) and Staphylococcus epidermidis (non-acid-
fast, light blue/purple). (Alan Schenkel, Peter Justice, and Erica Suchman, Colorado State University, Fort Collins, CO )
Endospore Stain
This stain is used to visualize bacterial endospores produced by members of the genera Bacillus and Clostridium. The
nature of the spore makes it impervious to most ordinary stains and staining methods, but, once stained it strongly resists
decolorization and counterstaining. In the Schaeffer-Fulton method, a primary stain with malachite green is forced into
the endospore by steaming the bacterial emulsion. Malachite green is water soluble and has a low affinity for cellular
material, so vegetative (actively dividing) cells may be decolorized with water. Vegetative cells are then counterstained
Image 5. Endospore stain of a Bacillus cereus culture using the Shaeffer-Fulton method and viewed at 1,000x total
magnification under an oil immersion lens. Vegetative cells of B. cereus are in red; endospores are in green. Due to the
age of the culture, endospores have been released from the cells. (Derek Weber and S. Finazzo, Broward Community
College–Central campus, Davie, FL)
Image 6: Encapsulated Enterobacter aerogenes stained with Anthony's capsule stain. (Gary E. Kaiser, The Community
College of Baltimore County, Catonsville Campus, Baltimore, MD)
Flagella Stain
This stain coats the thin bacterial flagella with heavy metals or other compounds to make them visible in the light
microscope. Once visible, the location and number of the flagella can be used diagnostically. The presence of flagella
varies with cultural conditions, so a negative result is not proof of a cell's inability to produce flagella. The demonstration
slide was prepared with a silver compound to coat the flagella and a red dye, basic fuchsin, to stain the cells.
Silver nitrate
Used to see bacterial flagella that are too slender to be seen with other staining techniques
Silver nitrate makes flagella appear larger than they are
Can be used to determine arrangement of flagella for identification.
Ex: Proteus vulgaris has peritrichous flagella
Image 7: Pseudomonas fluorescens stained with Presque Isle Cultures Flagella Stain. Arrows in the labeled view point to
the flagella. (Jay Mellies and Introductory Biology students, Reed College, Portland, OR)
Spirochete stain
Silver nitrate
Used to visualize slender spirochetes like Treponema pallidum
Image 9:Photomicrograph of skin biopsy showing secondary syphilis. Spirochete organisms are stained bright red by the
Treponema pallidum immunohistochemical stain. 400x magnification by Jerad M. Gardner,
References
1. Welcome to microbugz. Acid Fast Stain https://www.austincc.edu/microbugz/a...mplex%20lipids.
Watch Video 1: on heat fixing using a bacticinerator from Dr. G. Kaiser at URL: https://youtu.be/Rh0GrcTzjnU
Materials
Dropper bottle containing staining solution of methylene blue
Brightfield Light Microscope
Oil immersion
lens paper
Lab Procedures
A. Review Lab procedures for operating a Brightfield Light microscope
B. Preparation of a Bacterial Smear for Staining
Preparations of bacteria for staining can be made from growth on an agar plate or from a broth culture.
1. To prepare a slide from cells grown on an agar medium, first place a SMALL drop, a loopful works well, of water on a
clean grease-free slide. Next, with a sterile loop transfer a SMALL AMOUNT of the growth to the drop of water and rub the
loop around until the material is as evenly distributed as possible to form a just visibly turbid suspension. Spread the drop
over a small portion of the slide to make a thin film with lightly visible turbidity.
2. Prepare three separate smears, one each of Bacillus megaterium. Micrococcus luteus, and Saccharomyces cerevisiae.
3. Next, heat fix the slide by placing it on the slide warmer for 5 minutes. Put the sample side facing up and label with
your initials on one end of the slide. This process is called heat fixing the specimen to the slide. Its purpose is to bind the
specimen to the slide so that it does not wash off during staining. Killing the cells with heat fixation also increases their
permeability to the dyes used in staining.
Do not under-fix (the smear will wash off) nor over-heat (the cells will be ruptured or distorted) your specimen. The slide
should be warm to the touch, not hot. If you think you have heated the slide too much, do not touch the slide to your hand
to find out. Chances are, your suspicions are correct, and you will burn your hand with the hot glass. Instead, heat-fix the
slide for a shorter period of time next time, then test.
4. After cooling the specimen is ready for the simple stain.
E. Demonstration Slide
You have just prepared slides of Bacillus (a rod shape) and Micrococcus (a coccus). We have set up a slide to demonstrate
a third major cellular shape seen in bacteria. Spirillum volutans is a spiral shaped microorganism found in fresh water.
View the demonstration slide and record your observations in the Results page.
1 2/12/2022
5.1: Introduction to Enumeration of Bacteria
Learning Outcomes
Introduction to dilution theory
Estimate the number of microbes in a sample using serial dilution techniques and standard/viable plate counts
Enumeration of Bacteria
Often one needs to determine the number of organisms in a sample of material, for example, in water, foods, or a bacterial
culture. For example, bacterial pathogens can be introduced into foods at any stage: during growth/production at the farm,
during processing, during handling and packaging, and when the food is prepared in the kitchen (1). In general, small
numbers of pathogenic bacteria are not dangerous, but improper storage and/or cooking conditions can allow these bacteria
to multiply to dangerous levels (1).
Fecal contamination of water is another one of the ways in which pathogens can be introduced (1). Coliform bacteria are
Gram-negative non-spore forming bacteria that are capable of fermenting lactose to produce acid and gas. A subset of
these bacteria are the fecal coliforms, which are found at high levels in human and animal intestines. Fecal coliform
bacteria such as E. coli, are often used as indicator species, as they are not commonly found growing in nature in the
absence of fecal contamination (1). The presence of E. coli suggests feces are present, indicating that serious pathogens,
such as Salmonella species and Campylobacter species, could also be present (1).
Methods of Enumeration
Many approaches are commonly employed for enumerating bacteria, including measurements of the direct microscopic
count, culture turbidity, dry weight of cells, etc. In a microbiology lab, we frequently determine the total viable count in a
bacterial culture.
The most common method of measuring viable bacterial cell numbers is the standard or viable plate count or colony
count. This is a viable count, NOT a total cell count. It reveals information related only to viable or live bacteria. Using
this method, a small volume (0.1 - 1.0 mL) of liquid containing an unknown number of bacteria is spread over the surface
of an agar plate, creating a "spread plate." The spread plates are incubated for 24-36 hours. During that time, each
individual viable bacterial cell multiplies to form a readily visible colony. The number of colonies is then counted and this
number should equal the number of viable bacterial cells in the original volume of sample, which was applied to the plate.
For accurate information, it is critical that each colony comes from only one cell, so chains and clumps of cells must be
broken apart. However, many bacterial species grow in pairs, chains, or clusters, or they may have sticky capsules or slime
layers, which cause them to clump together. It is sometimes difficult to separate these into single cells, which in turn makes
it difficult to obtain an accurate count of the original cell numbers. Therefore, the total number of viable cells obtained
from this procedure is usually reported as the number of colony-forming units (CFUs).
A bacterial culture and many other samples usually contain too many cells to be counted directly. Thus, in order to obtain
plates, which are not hopelessly overgrown with colonies, it is often necessary to dilute the sample and spread measured
amounts of the diluted sample on plates. Dilutions are performed by careful aseptic pipetting of a known volume of sample
into a known volume of a sterile buffer or sterile water. This is mixed well and can be used for plating and/or further
dilution. If the number of cells in the original sample is unknown, then a wide range of dilutions are usually prepared and
plated. The preparation of dilutions and the calculation and use of dilution factors to obtain the number of microorganisms
present in a sample are important basic techniques in microbiology.
Method:
Aliquots from a stepwise or serial dilution of the original sample are spread on plates. Only a few of the plates following
incubation will contain a suitable number of colonies to count; those plated from low dilutions may contain too many
colonies to count easily while those plated from high dilutions may contain too few colonies or none at all. Ideally plates
Figure 1: Serial dilution series and plating. A wide series of dilutions (e.g. 10-2 to 10-8 ) is normally performed on the
sample culture and spread plates created from the dilutions. A number of spread plates is needed because the exact
number of live bacteria in the sample is usually unknown. Greater accuracy can be achieved by plating duplicates or
triplicates of each dilution.
Image 1: Picture of spread plates showing bacterial growth (Escherichia coli, 40 hours, 25°C) on five plates prepared
from a ten-fold dilution series. Care was taken to avoid spreading to the edges of the plates as it is more difficult to count
colonies along the edge of the agar. Note how many colonies are in the plate from the 10-1 & 10-2 dilution plates. These
plates have densely packed colonies, are too numerous to count, and most likely more than 300 CFUs. On the other
Calculations
In order to make the calculation of the number of cells/mL in the original sample less formidable, dilutions are designed to
be easy to handle mathematically. The most common dilutions are ten-fold and multiples of ten-fold. A 1/10 or 10-1
dilution can be achieved by mixing 1 mL of sample with 9 mL of sterile dilution buffer. A subsequent 1/10 dilution of this
first ten-fold dilution, made by mixing 1 mL of this first dilution with 9 mL of fresh sterile dilution buffer, would give a
total dilution of the original sample of 100-fold (1/10 X 1/10 = 1/100 or 10-1 X 10-1 = 10-2). Alternatively, a 100-fold
dilution can be made directly from the original sample by mixing 1 mL of sample and 99 mL of buffer. These dilutions can
be made in successive steps or a series to give a wide range for any given sample.
Once the dilution is made, an aliquot can be spread on an agar plate to create a spread plate. After incubation, the colonies
which arise can be counted and the number of cells (more precisely the number of colony-forming units or CFUs) in the
original sample can be calculated. For example in the image below, you did a serial dilution of a culture of the red
pigmented bacterium, Serratia marcescens and made a series of spread plates. In plate 1, this was the 10-1 dilution, in
plate 2 is the 10-2 dilution, and in plate 3 is the 10-3 dilution. After incubation, you count 241 colonies were present on the
plate 10-2 dilution.
Image 2: Three spread plates from serial dilutions. Image by Jackie Reynolds, Richland College, Dallas, TX.
Therefore, there were 241 X 102 CFU/mL in the original sample or expressed 2.41 x 104. To arrive at this final number
you only need to multiply the final number of colonies on the plate, 241, times the total dilution factor. The dilution factor
is defined as the inverse of the dilution. So in this case, the dilution factor is the inverse of 1/100 or 100. In other words,
the dilution factor is how many times the sample was diluted.
It usually works better to spread only 0.1 mL of a sample on a standard-size petri plate. If the above example were changed
such that 0.1 mL of a 100-fold dilution of the same sample was plated, there would ideally have been 24 colonies on the
plate. This number represents the number of CFU in only 0.1 mL of the dilution plated. Therefore, to calculate the CFU/ml
in the sample it is necessary to multiply the number of colonies on the plate by 10 (there are ten 0.1 mL units in 1.0 mL)
and then by the dilution factor (100) to arrive at the final answer: 24 CFU x 10 x 100 = 24000 or 2.4 x 104 CFU/mL.
The only way to understand dilution theory well is to practice it, so you should work practice problems until you feel
confident in using dilution factors and calculating CFU/mL in original samples. You should also be able to determine the
Watch Video 1 on how to perform a serial dilution using "pour plates" and the associated calculations:
Watch Video 1: Serial dilutions and pour plate technique. The example here using a "pour plate" technique to spread the
dilutions out instead of the "spread plate" discussed above, but the outcome of both techniques of spreading the dilution
sample out is the same. (10:48) Video by Microbial Zoo. URL:https://www.youtube.com/watch?v=nViO9Y4Yxfk
Watch video 2 on how to perform a serial dilution and make spread plates.
Watch Video 2: Serial dilutions with a bacterial sample and the 'spread plating' technique at NC State Microbiology labs.
(10:30) URL: https://youtu.be/IJcw4fRsYnU
References
1. Contributed by Joan Petersen & Susan McLaughlin Associate Professors (Biological Sciences and
Geology) at Queensborough Community College
5. Rotate the volume adjustment knob until the digital indicator reaches the desired volume, then place a disposable tip on
the shaft of the pipette (practice all three volumes min, int, max)
6. Press down on the plunger to the First Stop. (You will be able to push past this point, but there is enough resistance to
stop the movement if you try to be aware of it.)
7. Hold the pipette vertically and immerse the disposable tip into the sample. Use the colored water and the
microcentrifuge tubes provided to you.
8. Allow the plunger button to return slowly to its original position. Do not allow the button to snap up.
9. To dispense the sample: place the tip against the side wall of the receiving tube and push the plunger down to the first
stop. Wait 2-3 seconds, then depress the plunger to the second stop in order to expel any residual sample in the tip.
10. While the plunger is still pushed down, remove the pipette from the tube and allow the plunger to slowly return to its
original position.
11. Practice.
Micropipetting
Materials:
Cultures: Stationary phase broth culture of Serratia marcescens
Media: Dilution tubes of sterile water
Supplies: Nutrient agar plates, P-1000 Pipetman, sterile tips, Sterile L-shaped blue cell spreaders ("hockey stick” or
“spreader”)
Procedure:
A. Sample Dilution and Spread Plating
1. Label one plate on the bottom for each of the following dilutions: 10-5,10-6 , 10-7, 10-8,and 10-9. See Image 1.
2. Label the dilution tubes as follows: label the two tubes (Navy blue caps) containing 9.9 mL sterile water as 10-2 and
10-4; label the four tubes (Green caps) containing 9.0 mL sterile water as 10-5, 10-6, 10-7, and 10-8.
3. Carefully and aseptically remove 0.1 mL (100µL) of the Serratia marcescens culture and pipette it into the tube
marked 10-2. Mix the tube completely, being careful not to spill any of the contents. The vortex mixer will help you mix
the contents of the tube.
• You have now prepared a 10-2 (1/100) dilution. Why is it 10-2? Because 0.1 mL of undiluted culture was diluted into
9.9 mL of water, giving a total volume of 10.0 mL in the dilution tube. That makes this a 10-2 dilution (0.1/0.1 + 9.9 =
0.1/10.0= 1/100= 10-2).
4. Change the pipette tip, and take 100µL (0.1 mL) from the 10-2 dilution and pipette it into the next 9.9 mL dilution tube
marked 10-4. Mix well. You have now prepared a successive 1/100 dilution, resulting in 10-4 dilution of the original
culture (1/100 multiplied by 1/100 = 1/10,000 = 10-4, which is the same as 10-2 x 10-2=10-4).
5. Change your pipette tip again.
• Why keep changing pipettes? Because any fluid left in the pipette tip from the previous dilution will contain many
more cells per mL than any successive dilution and, if used, will grossly confuse the final results by indicating a higher
number of cells than were actually present in the original sample.
Now we will start doing a series of 1/10 dilutions. Set your Pipetman to 1,000µL and remove a 1,000µL (1.0 mL) aliquot
from the 10-4 dilution tube. Transfer it to the 9.0 mL dilution tube marked 10-5 and mix well. The original culture has now
been diluted 1/100,000, or 10-5 (10-4 x 10-1 = 10-5),
• You have just prepared a 10-1 (1/10) dilution of the previously diluted culture. Why is it 10-1? Because 1.0 mL of
diluted culture was further diluted into 9.0 mL of water, giving a total volume of 10.0 mL in the dilution tube. (1.0/1.0 +
9.0 = 1.0/10.0 = 1/10 = 10-1). Now you know why these series of dilutions are referred to as serial dilutions.
Continue your dilution series, as indicated in Image 1, through to the 10-8 dilution tube.
6. Using a new pipette tip, transfer 100µL (0.1 mL) of the 10-8 dilution onto the center of the agar surface of the plate
marked 10-9.
Consider why this is a 10-9 plate after you put 100uL (0.1 mL) of inoculum from the 10-8 dilution tube on it. Remember
that you are trying to determine the number of viable cells in each 1.0 mL aliquot of the original Serratia marcescens
sample, and you only put 10% of 1.0 mL (0.1 mL) on the nutrient agar plate.
Repeat with the rest of the dilutions, transferring 100µL (0.1 mL) of each dilution tube onto the appropriate agar surface,
using a new pipette tip between each dilution tube.
7. Spread plates: Using good aseptic technique, take one sterile blue L-shaped cell spreader out of the bag and reseal the
bag. Starting with the plate marked 10-9 , using the spreader, gently push the liquid inoculum applied to the center of the
plate, two or three times clockwise around the dish, then several times counterclockwise, turning the plate on the turn table
as needed to obtain complete coverage.
Continue with the rest of the spread plates using the same cell spreader. Make sure you are continuing to work backwards
(from 10- 9 , then 10-8 , then 10-7, then 10-6 etc.) from the most dilute suspension to the most concentrated suspension to
minimize the amount of carryover from plate to plate. Again, use good aseptic technique, work quickly, do not touch the
cell spreader on surfaces other than the agar plate you are using. If you accidentally “contaminate” the spreader by
touching a surface (table or other), then dispose of it and get a new spreader. We are trying to minimize the use of the
spreaders to reduce waste, but if it gets compromised, then replace it with a new sterile spreader.
Dispose of the cell spreader in the small orange biohazard bin on your bench when you are done with your spread plates.
Remember that the plates should be labeled as a ten-fold higher dilution than the dilution tube of the 0.1 mL sample being
plated. For example, 0.1 mL of the 10-6 dilution tube should be plated on the agar plate marked 10-7. Again, if you need
help visualizing this, see Figure 6-1.
It is generally desirable to make duplicate or triplicate plantings of each dilution and to average the resulting counts.
However, since your lab sample comes from the same stock culture, the class average should give an accurate enumeration
of the original stock culture.
8. After the spread plating, leave plates agar side down for at least 30mins. in order for the inoculum to absorb onto the
agar, then invert the plates and incubate at 30°C.
D. Watch this video on how to perform a serial dilution and make spread plates.
Watch Video 1: Dilutions and Plating at NC State Microbiology labs. URL: https://youtu.be/IJcw4fRsYnU
10-5
10-6
10-7
10-8
10-9
11
12
Class average:_____________________
4. Practice question
1/1000 135
10-6 48
99 9.9 x l06
10-5 1.0mL 28
10-6 1.0mL 7
2. How would you prepare a series of dilutions to get a final dilution of 10-10? outline each step.
3. When determining the number of bacteria from a sample using a viable plant count, why do you often have to do a
'serial' dilution of the sample first? Why don't you just plate out 1ml or 0.1ml of the sample on a spread plate?
4. Why is it called a "viable" plate count? what does that mean?
1 2/12/2022
6.1: Introduction to Oxygen Requirements
Learning Outcomes
Recognize the effects of Oxygen on bacteria
Explain the various oxygen requirements of the microbes, observe and interpret the growth of microbes in
thioglycollate agar deep media
Discuss methods of culturing anaerobic bacteria
Bacteria and many microorganisms are very sensitive to oxygen concentrations. Some will only grow in its presence and
are called obligate aerobes. Facultative aerobes will grow either aerobically or in the absence of oxygen (anaerobic
conditions), but they generally do better with oxygen. Aerotolerant anaerobes don't require oxygen, but can grow in its
presence, while strict obligate anaerobes cannot use oxygen and cannot grow or survive in its
presence. Microaerophiles use oxygen, but at lower concentrations than atmospheric oxygen levels (which is ~20%).
One can determine a bacterium's oxygen requirements by cultivating them in a special medium called thioglycollate agar
tubes. The bottom of the tube of medium is kept anaerobic by cystine and thioglycollic acid, which chemically react with,
and tie up any oxygen that diffuses in. Any un-reacted oxygen in the tube will be indicated by resazurin, a dye that turns
pink in the presence of oxygen. It is common for the top centimeter or so to be pink. One can inoculate a thioglycollate
tube with your bacterium and observe where the bacterium grows in the tube to determine its oxygen requirements (see
Image 1)
Image 1: Microbial oxygen requirements determined using thioglycollate agar tubes. Green dots represent
bacterial colonies within in the agar or on its surface. The surface of the agar tube is directly exposed to atmospheric
oxygen, and will be aerobic. The oxygen content of the thioglycollate medium decreases with depth until the medium
becomes anaerobic towards the bottom of the tube.
Watch Video 1: explanation on how thioglycollate media works and examples. (9:33)
URL: https://youtu.be/AJG18sQd8mU
Watch Video 2: how to set up an anaerobic jar. The process is similar for an anaerobic box. (3:03)
URL: https://youtu.be/aFDYx-7ceS8
Learning Objectives
Identify the 3 major categories of microbes based on oxygen requirements.
Learn different ways to culture anaerobic bacteria.
Image 1: On the left is a GasPak jar, with a GasPak generator envelope inside. The
environment is 0% O2. On the right is a candle jar with reduced
atmospheric O2 concentrations and CO2 .
The newer anaerobic system (image 2) consists of a plastic container or box (for the
agar plates) and a GasPak paper generating sachet. The sachet contains ascorbic acid
and activated carbon which reacts on exposure to air, when removed from the enclosed
envelope. Oxygen is rapidly absorbed and CO2 is produced. When the GasPak paper
sachet is placed in a sealed plastic pouch, this reaction will create ideal atmospheric
conditions for the growth of anaerobes—anaerobic within 2.5 hours.
Because a GasPak jars and boxes looks the same, whether it has oxygen inside or not, an
indicator strip, containing methylene, is included in the jar. Methylene blue is blue when oxidized, colorless when reduced.
The carbon within the pouch reacts with free oxygen in the jar, producing 10-15% CO2.
Quite a few human pathogens are strict anaerobes, exemplified by the bacillus-shaped genera---Gram-
negative Bacteroides, Bacillus (anthracis), and Gram-positive Clostridium (tetani, botulinum).
Aerotolerants are anaerobes that can grow in the presence of O2 (compared to the strict anaerobes which would likely
die), but they do not use it. And last, but very common, are the facultative anaerobes which prefer to use O2 when present
but will grow without it.
Another way to culture and grow anaerobes is the use of reduced media--media without oxygen. Thioglycollate
broth/agar has a reducing agent in it---the chemical thioglycollate---which binds any free oxygen within the medium.
Note
Oxygen will permeate the broth when this medium sits around for a while.
Check for the pink color: if so, boil the broth for 5 minutes (removes the oxygen).
growth is indicated by gray area
PROCEDURE
Thioglycollate broth
1. The thioglycollate broth should be either boiled first before
inoculation OR recently made so that the oxygen content is
very low. (Your instructor will tell you if it needs to be boiled).
2. Inoculate a tube of thioglycollate broth with your unknown
bacterium: make sure that the loop or needle goes down to the BOTTOM of the broth (do not get metal
holder in the sterile broth).
3. Incubate at 25 or 37 degrees C as directed.
Thioglycollate broth
Determine WHERE the most amount of growth occurs in the column of liquid---the top, the bottom, top to bottom.
DO NOT SHAKE IT! How can you determine if the bacterium is aerobic, anaerobic, or facultatively anaerobic?
Learning Outcomes
Recognize the cardinal temperatures of growth for bacteria
State the conditions for classification of psychrophiles, mesophiles, thermophiles, and hyperthermophiles
Recognize the pH requirements for bacteria
Recognize the osmotic requirements for bacteria
Microorganisms require a temperature growth range dictated by the heat sensitivity of its cellular components. As a
result, microbial growth has a characteristic temperature dependence with distinct cardinal temperatures---the minimum,
optimum, and maximum, temperatures at which it can grow. The optimum temperature is usually correlated to its natural
habitat.
Environmental Requirements: pH
The pH is another environmental condition that dictates microbial growth. pH impacts the activities of enzymes and
each microbial species has a pH growth range. Acidophiles have a growth range between pH 0.0-5.5,
neutrophiles grow btween 5.5 and 8.5, while alkalophiles do best between 8.5-11.5 (or higher). Generally, different
microbial genera have characteristic pH optima ranges. The majority of bacteria are neutrophiles while molds and
yeasts tend to prefer slightly acidic environments with a pH range of 4-6. Many bacteria produce acids as part of their
metabolism, and this can lower the pH of their environment. One excellent example of this are the Lactic Acid
Bacteria (LAB); a large and diverse group of Gram-positive bacteria that produce lactic acid as the major end product
of the fermentation of carbohydrates. The lactic acid can inhibit the growth of pathogenic and food spoilage
microorganisms in food. Thus the LAB play a significant role in food fermentation, contributing to a wide variety of
fermented products (ie. cheese, yogurt, meat, fish, fruit, vegetable and cereal products). Their breakdown of various
carbohydrates, proteins, and lipids contribute to the flavor, texture and nutritional value of the fermented foods.
Bacteria Growth
Viruses are obligate intracellular parasites that multiple within the host cytoplasm. Bacteriophages are viruses which
infect bacteria. PHAGE (as in phagocytosis) means "to eat", and generally refers to a virus. Viruses multiply within host
cells and rely upon the host's metabolic machinery for replication.
Most bacteria have phages that are able to parasitize them. In fact, the ability to be infected with a known phage type is
used to identify some strains of bacteria (like Staph), known as phage typing. As the virus infects bacterial cells that it has
been mixed with, the lytic infection destroys the bacteria. The bacteria have been poured into what is called a bacterial
lawn on the agar plate. As the surrounding cells are infected and killed by the released viruses, a clear spot on the agar---in
the bacterial lawn--develops, called a plaque.The plaques can be counted and the number of virus particles or virions in
the original specimen, can be quantified as viruses/ ml or plaque-forming units/ml (PFUs).
One common phage is called "T4" and it is capable of infecting its host, Escherichia coli. This type
of bacteriophage is called a "coliphage. " Viruses have high specificity for its host, and will only infect a specific
bacterium host.
General procedure
1. The phage specimen you will use is already diluted to 1/ 1000, and you will dilute further.
2. Bacteria and phage are mixed together in tubes of soft agar. The mix is incubated in the water bath.
3. After incubation the mix is added to the soft agar and poured over the tryptone agar plates.
4. Be SURE to mix the dilutions.
5. Change pipettes between dilutions.
6. Each table will use a different combination of a phage and an E. coli host.
Set up 5 saline (0.85% NaCl) dilution tubes labeled 10-4, 10-5,10-6, 10-7, and 10-8. Into each tube, place 9ml pf into which
you will dilute the viral solution. You will be making 1/10 dilutions.
1. Starting with the 10-3 dilution of the virus that you picked up (or were given by your instructor), transfer 1 ml to the
dilution tube marked 10-4 and mix.
2. Make 4 more dilutions out to 10-8.
3. Into 6 microtubes, add 100 microliters of each viral dilution, plus 300 microliters of E. coli. Let sit at room
temperature for 10 minutes while the virus infects the bacteria. Mix these well.
4. Take the tubes over to the water bath and transfer the entire contents of E. coli - phage tubes into 6 soft agar tubes,
using a sterile plastic transfer pipet. Mix well. KEEP SOFT AGARS INSIDE OF WATER BATH SO THEY DO
INTERPRETATION
Image 2: Determination of bacteriophage T4 titer by serial dilution and plating on Escherichia coli B host. These series of
plates show viral plaques--note the round, clear zones on the agar bacterial lawn on each plate (see arrow pointing to
one). Image by Anh-Hue T. Tu, Georgia Southwestern State University, Americus, GA.
1. Lay the 6 plates right side up, from lowest dilution towards highest dilution.
2. Pick each plate up, hold it up to the light, and determine which one has between 30 - 300 plaques (you can also use the
Quebec colony counters---good backlighting!)
3. Get an accurate count of that plate. Fill in the formula for viral counts.
Procedure
Thioglycollate broth
1. The thioglycollate broth should be either boiled first before inoculation OR recently made so that the oxygen content is
very low. (Your instructor will tell you if it needs to be boiled).
2. Inoculate a tube of thioglycollate broth with your unknown bacterium: make sure that the loop or needle goes down to
the BOTTOM of the broth (do not get metal holder in the sterile broth).
3. Incubate at 25 or 37 degrees C as directed.
Watch video 1: This procedure is showing you how to inoculate a deep stab agar slant. A similar procedure can be used
for inoculating thioglycollate tubes, except that the media is semi-solid agar and there is typically no slant, so you don't
TSA plates
Compare the presence/absence of growth, as well as the quantity of growth on the 3 plates. Determine whether aerobic,
anaerobic, or facultatively anaerobic.
To the right:
A is an facultative anaerobe
B is an aerobe (microaerophilic)
C is an anaerobe
Thioglycollate broth
Determine WHERE the most amount of growth occurs in the column of liquid---the top, the bottom, top to bottom. DO
NOT SHAKE IT! Can you determine if the bacterium is aerobic, anaerobic, or facultatively anaerobic?
Contributors
1. Jackie Reynolds, Professor of Biology (Richland College)
Plaque Assay
Knowing how to determine the number of microorganisms in a sample is extremely important in microbiology, and
requires accurate pipetting, aseptic technique, and calculation skills. In this exercise, you will again prepare dilutions and
enumerate the microorganisms in a sample. Although the principles are the same, you will be enumerating viruses instead
of bacteria and conserving on materials by using micropipettes and smaller volumes in your dilutions.
The number of viruses in a sample can be determined by direct count using electron microscopy or by determining the
number of infectious virus particles using a plaque assay. Viruses are obligate intracellular parasites. Therefore, they
require a host cell in which to grow. Some viruses lyse the cells in which they have replicated while others appear to cause
little cell damage. A plaque assay can be used to enumerate viruses that lyse their host cells. In a plaque assay the host
cells and virus are incubated together for a short time to allow the virus to attach to and enter the host cell. Then the
mixture in plated within a semi-solid agar. This semi-solid agar is poured onto a "bottom agar" that serves to supply
adequate nutrients for the host cell. At the end of one cycle of virus replication a cell infected with a single virus particle
will lyse, releasing hundreds of new viruses. In the semi-solid medium these newly released viruses can only infect
neighboring cells; after a second cycle of replication these neighboring cells will be lysed. Those cells that escape infection
will continue to grow. After 24 to 48 hours plates that were not infected with a virus will contain a confluent layer of cells
(they will resemble the TNTC plates of the preceding exercise). Those plates that were infected with several hundred
viruses may actually appear clear- the viruses will have infected and lysed all of the host cells. Those plates that contain an
intermediate number of viruses will have plaques, clear or partially clear circular areas in an otherwise turbid background
of cellular growth. Each plaque represents the result of one infectious virus, called a plaque forming unit, or PFU. Many
animal and bacterial viruses can be enumerated using a plaque assay. Bacterial viruses are also referred to as phage; in this
exercise you will be using a well-characterized bacterial phage, T4, and its host cell, Escherichia coli.
In this exercise you will also be setting up your own dilution tubes and using micropipetters in this laboratory. Your
laboratory instructor will go over the proper use of these pipetters and maintenance of aseptic technique.
Materials:
Cultures Escherichia coli strain B culture
Phage sample T4
Media 4 petri plates containing "bottom agar"
4 tubes containing 5 mL of "top agar" - these tubes will be in the 50°C water bath
Supplies Sterile Tryptic Soy broth diluent and 10 sterile microcentrifuge tubes
Micropipetters and sterile tips
Procedure:
A. Sample Dilution and Plating
1. Prepare 10-fold serial dilutions of the virus through 10-6
2. Label your remaining 4 sterile tubes 10-4, 10-5, 10-6, and 10-7, and pipette 100 µL from the appropriate phage dilutions
into these labeled tubes (use a new pipette tip for each transfer).
3. Add 100 µL of the Escherichia coli culture to each of the 4 labeled tubes. (Use the same pipette tip; add Escherichia
coli to the tube with the most diluted phage first and work back.)
4. Incubate bacteria with virus for 10 minutes to allow for adsorption (attachment) of the virus to the bacteria.
5. While you are waiting, label the agar plates with your name, lab section, and the virus dilution that will be plated on it:
one plate for each 10-4, 10-5, 10-6, and 10-7. Reset the micropipette for 200 µL.
6. After the 10 minutes, get the tubes of top agar from the 50°C water bath; working quickly, wipe off the tubes. You may
want to get one top agar tube at a time so you won't have to rush; top agar solidifies within a few minutes once it is
removed from the 50°C bath.
7. Pipette 200 µL from a labeled tube into a top agar tube, vortex for about 2 seconds, pour onto the appropriately labeled
bottom agar plate. Do this for all four labeled tubes.
8. Allow the top agar to solidify (about 5 minutes) before moving the plates. Invert the plates and incubate at 30°C.
10-4
10-5
10-6
10-7
Student pairs Dilution with 30-300 plaques Number of plaques on plate PFU/mL in original T4 phage
sample.
10
11
12
13
Class Average:
1 2/12/2022
7.1: Introduction to Biochemical Tests Part I
Learning Outcomes
Observe and interpret the fermentation reactions of representative bacteria in phenol red sugar broths, distinguish
between respiration and fermentation, discuss the conditions in which these reactions occur.
Observe and interpret sugar fermentation and hydrogen sulfide formation in TSI agar slants, discuss the purpose of
critical ingredients in TSI agar slants, distinguish between different sugar fermentations, interpret TSI reactions.
Learn about the role of extracellular enzymes in bacteria, observe the hydrolysis of casein hydrolysis
A. Carbohydrate Fermentation
Fermentation is a metabolic process that some microorganisms use to break down substrates such as glucose and other
sugars when O2 is not available or could not be used by the microorganism. Fermentation includes the reactions of
glycolysis (where a single molecule of glucose is broken down into 2 molecules of pyruvate), as well as additional
reactions that produce a variety of end products (acids, alcohols, gases). The end products are characteristic of individual
bacterial species. Keep in mind, microbes are very versatile, the fermentation substrate does not have to be sugars, it can
include even unusual compounds like aromatics (benzoate), glycerol (sugar-alcohol), and acetylene (hydrocarbons)!
Much of the original energy in the substrate remains tin the chemical bonds of organic end products, like lactic acid or
ethanol. For example, one fermentation waste product is ethanol, its got so much stored energy it can be used in gasoline
solutions to be combusted/burned to release that energy stored in its chemical bonds.
Note that fermentation is mainly a mechanism for regenerating NAD+ when respiratory process do not occur.
Fermentation also tends to produce waste products that can accumulate in the extracellular environment. By contrast, the
waste left over after ATP production by aerobic respiration are limited to CO2 and H2O. There can be numerous end
products from fermentation, many of which is useful for us, but not necessarily the microbes. We use many fermentation
products--as diverse as antibiotics, alcohols, and a variety of foods. Microbes such as yeast and bacteria are genetically
engineered to produce valuable fermentation products.
Image 1: Fermentation Reactions Produced by Escherichia coli in Phenol Red Sugar Broths Containing Dextrose,
Sucrose, and Lactose sugars. Image by Janie Sigmon, York Technical College, Rock Hill, SC.
In many metabolic tests, end products are produced that change the pH of the medium. To measure this pH change, pH
indicators (chemicals that change color depending on pH) are included in the medium. Some common pH indicators are
phenol red, bromocresol purple, and bromothymol blue. Each pH indicator has a range of pH values over which it changes
color (see below).
pH Indicator
phenol red < pH 6.8 = yellow pH 6.8 – 7.4 = red pH >7.4 = pink/magenta
bromothymol blue < pH 6.0 = yellow pH 6.1 – 7.5 = green pH >7.5 = blue
Table 1: pH indicators
Watch Video 1: how to perform phenol red sugar tests. Video by Microbial zoo (3:40).
URL: https://youtu.be/W8JWInjlXqQ
Image 3: Proteus mirabilis in a triple sugar iron (TSI) slant. This organism ferments only glucose, indicated by the red
coloring of the agar. The slant is red due to depletion of glucose and the subsequent digestion of proteins in the agar.
There is a large carbon dioxide bubble in the bottom right area of the tube, and the black precipitate indicates hydrogen
sulfide was produced. Image by Diane Hartman, Baylor University, Waco, TX.
Image 4: Proteus vulgaris in a triple sugar iron (TSI) slant. This organism ferments glucose and sucrose. Acid causes the
phenol red indicator in the agar to turn yellow. There is a small carbon dioxide bubble in the bottom right area of the tube.
The black precipitate indicates hydrogen sulfide was produced. Image by Diane Hartman, Baylor University, Waco, TX.
Image 5: Alcaligenes faecalis in a triple sugar iron (TSI) slant. This organism does not ferment sugars so the medium
remains red (no acids are produced in the slant or butt). The slant becomes a deeper shade of red indicating the organism
Watch Video: how to inoculate & interpret a TSI agar slant. Video by MCCC Microbiology (1:35)
URL: https://youtu.be/FuOcN3wB0VM
C. Extracellular enzymes
An exoenzyme, or extracellular enzyme, is an enzyme that is secreted by a cell into the environment and functions outside
of that cell. Exoenzymes are produced by both prokaryotic and eukaryotic cells. Most often these enzymes are involved in
the breakdown of larger macromolecules. The breakdown of these larger macromolecules is critical for allowing their
smaller components to pass through the cell membrane and enter into the cell. Bacteria and fungi also produce exoenzymes
to digest nutrients in their environment, and these organisms can be used to conduct laboratory assays to identify the
presence and function of such exoenzymes. Some pathogenic species also use exoenzymes as virulence factors to assist in
their spread.
i. Casein Hydrolysis
Some bacteria secrete extracellular enzymes called proteinases that break down proteins. Milk contains large proteins
called casein. Some bacteria secrete caseinases that break down casein outside of the bacterial cell so the smaller products
(e.g., amino acids) can be transported inside the cell and further metabolized.
Milk agar (which contains powdered milk) is used to detect the presence of bacterial caseinases. This medium (Image 6)
is cloudy because when milk is mixed with agar, the casein forms a colloid through which light cannot pass. The presence
of caseinases can be detected by observing a clearing in the agar around the bacterial growth, which indicates that the
caseins have been broken down into transparent end products (amino acids and peptides), which are then taken up by the
cells (image 7).
To test for the presence of alpha amylase, a starch hydrolysis test can be performed. Gram's iodine can be used to
indicate the presence of starch, when it contacts starch, it forms a blue to brown complex. If the starch has been broken
down/hydrolyzed, then there is a clear area that appears in the medium upon addition of Gram's iodine. This clearing zone
indicates the presence of alpha amylase.
Image 9: Growth of Bacillus subtilis on a starch agar plate before the addition of iodine solution (A) and after the addition
of iodine solution (B). After the addition of iodine, the clearing surrounding the bacterial growth indicates starch
hydrolysis. Image by Archana Lal, Independence Community College, Independence, KS.
A. Catalase Activity
Byproducts of aerobic metabolism include two toxic compounds: superoxide free radicals (O2 -) and hydrogen peroxide
(H2O2). These toxic compounds can cause intracellular damage, such as damage to DNA, lipids, and proteins. To remove
these compounds, cells produce enzymes to break them down. Cells can convert superoxide free radicals to hydrogen
peroxide by using the enzyme superoxide dismutase (SOD); catalase breaks down hydrogen peroxide into water and
oxygen.
A simple test to determine if bacteria produce catalase is to add hydrogen peroxide to bacteria on an agar slant or to
bacteria spread on a slide (image 1). If catalase is present, the hydrogen peroxide will be broken down into water and
oxygen gas, resulting in the production of bubbles (+ test). This test does not require any special type of medium, however
it should never be performed on organisms that have been grown on blood agar (a medium that contains blood). This is
because there is a catalase activity in blood that would produce a false positive result. Most aerobic and facultatively
anaerobic organisms produce SOD and catalase (note: some species use peroxidase rather than catalase to break down
hydrogen peroxide). Obligate anaerobes lack these enzymes, which is why they cannot survive in an atmosphere
containing oxygen. However, some of them have modified versions of these enzymes to deal with any possible exposure to
oxygen. The archaeon, Pyrococcus furiosus, is an obligate anaerobe that lives on and near hydrothermal vents. Certain
segments of these habitats are anoxic and P. furiosus occupies these niches. Some anaerobes have a superoxide-reducing
system based on a different enzyme, called superoxide reductase (SOR), which reduces O2− rather than dismutating it.
B. Oxidase test
Cytochrome oxidase, also known as complex IV, is the terminal, or final, enzyme of the electron transport
system/ETS (this does not include ATP synthase). Cytochrome oxidase is a transmembrane molecule found in the
mitochondria of eukaryotes and in the cellular space of aerobic prokaryotes. This molecule is a proton pump that plays a
vital role in producing energy, in the form of ATP, via the ETS. In the last steps of the energy production process,
cytochrome oxidase oxidizes the waste products from the end of the energy making process, converting reactive species,
H+ and dioxygen (O2), to a more stable molecule, water (H2O).
The oxidase test is a key test to differentiate between the families of Pseudomonadaceae (ox +)
and Enterobacteriaceae (ox -), and is useful for speciation and identification of many other bacteria, those that have to use
oxygen as the final electron acceptor in aerobic respiration. The enzyme cytochrome oxidase is involved with the reduction
of oxygen at the end of the electron transport chain.
A cytochrome c oxidase test utilizes a special reagent called oxidase reagent, which is a 1% solution of the chemical
tetramethyl-para-phenylenediamine (TMDPD) dihydrochloride. The reduced form of this reagent is colorless, but donates
electrons to the cell’s cytochrome c oxidase forming an oxidized colored form.
Oxidase positive bacteria change the color of the reagent from colorless to colored and finally black.
Image 2: Oxidase test on filter paper. A positive oxidase result given by Pseudomonas aeruginosa (left) is indicated by a
purple color. A negative oxidase result given by Escherichia coli (right) is indicated by the lack of color change. Both
organisms were rubbed onto a filter that was dipped in oxidase reagent and allowed to dry. Image by Laura Cathcart,
University of Maryland, College Park, MD; Sabrina Kramer, University of Maryland, College Park, MD; Patricia Shields,
University of Maryland, College Park, MD.
Image 3: Oxidase test on an agar plate with bacterial colonies. This close up view of an agar plate has a mixed culture
of oxidase-positive Vibrio cholerae, indicated by purple colonies, and oxidase-negative Escherichia coli, indicated by lack
of color change (they are the white colonies), demonstrate how the plate oxidase test differentiates between the two. In
this test, the oxidase reagent was added directly to the colonies which were grown on trypticase soy agar at 37°C for 24
hours. Image by Laura Cathcart, University of Maryland, College Park, MD; Sabrina Kramer, University of Maryland,
College Park, MD; Patricia Shields, University of Maryland, College Park, MD.
Oxidase test is most helpful in screening colonies suspected of being one of the Enterobacteriaceae (all negative) and in
identifying colonies suspected of belonging to other genera such as Aeromonas, Pseudomonas, Neisseria, Campylobacter,
and Pasteurella (positive).
Watch Video 2: how to perform an oxidase test. (3:23) Video by URMICRO1. URL: https://youtu.be/7Aa1xO2cC1M
Image 3: Sulfur-indole-motility (SIM) test results from various microbes. From left to right: (A) Escherichia coli,
(B) Staphylococcus aureus, (C) Salmonella arizonae, (D) Enterobacter aerogenes, and (E) Proteus vulgaris. After addition
of Kovács reagent, a pink ring at the top of the tube indicates a positive indole result (A and E). Blackening of the media
indicates hydrogen sulfide production (C and E). Growth feathering away from the stab line creating a cloudy appearance
in the media indicates motility (A, C, D, and E). Growth strictly along the stab line indicates a nonmotile organism (B).
Image by Tasha L. Sturm, Cabrillo College, Aptos, CA.
Note
All media that you inoculate today will be incubated until the next lab, when you will analyze your results .
A. Carbohydrate Fermentation
Each student: 1 tube each of the following broths: Lactose + phenol red (green cap), Sucrose + phenol red (yellow cap),
Glucose + phenol red (red cap)
Instructions: Choose 1 of the following bacteria: Proteus vulgaris, Escherichia coli, Bacillis subtilis, or Streptococcus
faecalis (each person at the table should choose a different species)
Inoculate the 3 types of fermentation broth with your chosen bacteria. Prior to inoculating the broths, make note of any
small bubbles that might be present in the Durham tubes, so these are not read as evidence of gas formation during
fermentation.
1. Inoculate your TSI slant/deep with your assigned bacteria using a needle by stabbing fully into the butt ONCE and only
ONCE.
2. Then, streak across the slant WITHOUT re-dipping your needle in your plate. KEEP CAPS LOOSE and place it on the
rack at the end of the table to be incubated at 37°C
D. Catalase Activity
Procedure:
1. Transfer a loopful of cells of the species to be tested onto a microscope slide. Bacillus megaterium should be used as a
positive control and your unknown should be tested.
2. Add a drop of hydrogen peroxide.
3. Look closely for the evolution of bubbles.
E. Oxidase test
Procedure for BD BBL DrySlide: (each pair of students need 1 slide)
1. Open the BD BBL DrySlide Oxidase pouch and remove a slide by grabbing the edge of the slide, do not grab paper
slide areas. After removing a slide, fold the top of the pouch over and seal tightly with a self-adhesive sticker (provided).
2. Using a sterile toothpick, pick a portion of the colony to be tested and apply the growth on ONE quadrant of the dry
slide. To ensure a proper reaction, spread the inoculum on the slide reaction area to a 3-4mm size. Roll the toothpick over
the slide reaction area to transfer the cells.
3. On one oxidase slide, there are 4 quadrants: perform this test on Bacillus megaterium as a negative control,
Pseudomonas fluorescens as a positive control, your unknown, and your partner’s unknown. Examine the reaction area for
appearance of a dark purple/blue color within 20sec. Disregard color development after 20 sec.
For Gibson Bioscience Swabs: (1 swab per sample used)
1. Open the Gibson Bioscience Swabs package, ensuring that you open only at the end where you can grab the handle of
the swab. Touch the swab to a (control(s) or unknown sample) colony on your plate. Examine for purple/blue coloration
after 10 sec. Disregard any color development occurring after 60 sec.
A. Carbohydrate Fermentation
Observe the results of your own carbohydrate fermentation test, as well as the tests done by your table partners. You can
compare your inoculated tubes with the negative controls in the front of the class at the instructor’s table. Record the
results in the table below.
The following convention is used when noting the results of fermentation experiments.
A = acid
G = gas
AG means that both acid and gas are present
If neither acid nor gas is present, you can write “negative”
Proteus vulgaris
Escherichia coli
Bacillus subtilis
Streptococcus faecalis
B. TSI
Bacterium Glucose Lactose Sucrose Gas production H2S production
C. Casein Hydrolysis
Observe your casein plate. It is helpful to hold it up to the light so you can detect the clear zones. Make a drawing of your
results the circle below. Indicate the location of the bacterial growth and draw any clear zones that are present. Record your
results in the table below.
A. Enterobacter aerogenes
B. Bacillus subtilis
D. Catalase Activity
Add a dropper full of H2O2 to the surface of the slant. Record your results below.
Bacillus megaterium
Unknown
E. Oxidase
Bacteria Color Oxidase Result (+/-)
Bacillus megaterium
Pseudomonas fluorescens
F. SIM Tubes
NOTE: Be sure to look on the surface of the agar as well as within the deep stab for evidence of motility.
A. SIM agar deep tubes
Klebsiella aerogenes
Staphylococcus epidermidis
Proteus vulgaris
Unknown
1 2/12/2022
8.1: Introduction to Bacterial Identification using Culture Media
Learning Outcomes
Identify and describe culture media for the growth and identification of bacteria, including examples of selective
and/or differential media.
typhi, coli, proteus, bacteria, cultured, bismuth, sulfite, agar, 48hrs, incubation
Differential media makes it easier to distinguish colonies of your desired microorganism from other colonies growing on
the same plate. Blood agar (which contains red blood cells/RBCs) is a medium often used to identify bacterial species that
destroy RBCs. These species, such as Streptococcus pyogenes, that causes strep throat, will show a clear ring around their
colonies where they have lysed the surrounding blood cells.
Image 2: Normal Upper respiratory flora mixed with Streptococcus species. The presence of beta-hemolytic colonies
(clear zones around small colonies) indicates the possibility of Streptococcus pyogenes infection. Image by Rebecca
Buxton, University of Utah, Salt Lake City, UT.
Image 3: Mannitol salt agar inoculated with Staphylococcus aureus on the left side of the plate and showing
fermentation of mannitol (yellow medium) and inoculated with Streptococcus durans on the right side of the plate, which
shows no growth (no colonies visible, medium remains reddish pink). Image by Anne Y. Tsang and Patricia Shields,
University of Maryland, College Park, MD.
Watch Video 1: Mannitol Salt Agar explained and showing plate examples. Video by Dr. Gary Kaiser (CCCB ). (1:38)
URL: https://youtu.be/kG1_Tf5Vpc0
Image 4: MacConkey agar plate inoculated with the Gram-negative lactose fermenter (pink colonies/streak) Escherichia
coli and the Gram-negative non-lactose fermenter (off yellow colonies) Serratia marcescens. Image by David Miller and
Patrick Hanley, Hartwick College, Oneonta, NY)
MacConkey Agar
Watch video 2: MacConkey agar explained and showing plate examples. Video by Dr. Gary Kaiser (CCCB ). (4:32)
URL: https://youtu.be/yInQ9jApAlU
Image 5: Eosin-methylene blue ( EMB) agar plate inoculated with Escherichia coli (a Gram-negative coliform bacterium)
showing good growth of dark blue-black colonies with metallic green sheen indicating vigorous fermentation of lactose
and acid production which precipitates the green metallic pigment. Image by Naowarat Cheeptham, Thompson Rivers
University, Kamloops, BC, Canada.
Image 8: Enterobacter aerogenes on Hektoen enteric agar. Note the yellow-orange colonies, indicating the
fermentation of at least one of the carbohydrates present in the medium. The lack of black colonies indicates no H2S
production. The orange haze around the colonies is due to the precipitation of the bile salts by the organism. The
appearance of E. aerogenes on Hektoen enteric agar is typical of most nonpathogenic enteric Gram-negative rods. Image
by Jan Hudzicki, University of Kansas Medical Center, Kansas City, KS.
Bacterial Identification
The identification of microorganisms is an important part of what many microbiologists do. As you can imagine, clinical
microbiologists would need to identify the pathogen that is causing disease in patients. A microbial ecologist is interested
in what microbes are contributing to environmental change or they might want to identify new species in the field. Perhaps
you are working in a lab and have a contaminant and want to know where it is coming from and what it is. We have
already learned some ways to identify microbes, in the last module, you learned about the many biochemical tests
available to microbiologists that aid in their identification of bacteria. We’ll continue with some of these biochemical tests,
focusing on ones that are capable of combining multiple biochemical tests into one, and the use of selective and differential
media to isolate and identify bacteria. Then we’ll move into bacterial genetics and how we can use molecular methods to
identify bacteria.
Enteropluri/Enterotubes
A number of techniques can be used for the identification of specific species and subspecies
of Enterobacteriaceae. Speciation is important because it provides data regarding patterns of susceptibility to
antimicrobial agents and changes that occur over a period of time. It is also essential for epidemiological
studies such as determination of nosocomial infections and their spread.
In an effort to simplify the speciation of the Enterobacteriaceae and reduce the amount of prepared media and
incubation space needed by the clinical lab, a number of self-contained multi-test systems have been
commercially marketed. Some of these multi-test systems have been combined with a computer-prepared
manual to provide identification based on the overall probability of occurrence for each of the biochemical
reactions. In this way, a large number of biochemical tests can economically be performed in a short period of
time, and the results can be accurately interpreted with relative ease and assurance.
The EnteroPluri-Test /Enterotube is a self-contained, compartmented plastic tube containing 12 different agars
(enabling the performance of a total of 15 standard biochemical tests) and an enclosed inoculating wire. After
inoculation and incubation, the resulting combination of reactions, together with a Computer Coding and
Identification System (CCIS), allows for easy identification. The various biochemical reactions of the
EnteroPluri-Test and their correct interpretation are discussed below. Although it is designed to identify
members of the bacterial family Enterobacteriaceae, it will sometimes also identify common biotypes
of Pseudomonas and other non-fermentative Gram-negative bacilli. It does not identify Pseudomonas
aeruginosa.
IDENTIFYING MEMBERS OF THE ENTEROBACTERIACEAE WITH THE ENTEROPLURI-TEST
The EnteroPluri-Test contains 12 different agars that can be used to carry out 15 standard biochemical
test. Interpret the results of your EnteroPluri-Test is based on a coding chart included with the test.
The enterotube is a self-contained, compartmented plastic tube containing twelve different media that allow determination
of 15 biochemical reactions (glucose, gas production from glucose, lysine decarboxylase, ornithine decarboxylase,
hydrogen sulfide (H2S), indole, adonitol, lactose, arabinose, sorbitol, Voges-Proskauer (VP), dulcitol, phenylalanine
deaminase (PA), urea, and citrate). The enclosed inoculating wire allows inoculation of all compartments in one step from
one or a few single colonies of your unknown microorganism. The resulting combination of enterotube reactions, together
with other metabolism tests can help you to identify unknown organisms.
End products of bacterial fermentation of glucose are either acid, or acid and gas. Gas production will be indicated by the
definite and complete separation of the wax overlay from the surface of the glucose chamber. The glucose chamber
medium is covered with wax to provide anaerobic conditions to allow detection of gas formation. Fermentation of
adonitol, lactose, arabinose, and sorbitol will also result in formation of acidic end products indicated by a change in color
of indicator present in the medium from red to yellow. Any sign of yellow is interpreted as a positive reaction; red should
B. Urease
Many bacteria are able to use urea as a nitrogen source by splitting it into ammonia and carbon dioxide through the
hydrolysis reaction catalyzed by the enzyme urease:
O
ǁ urease
NH2 — C—NH2 + H20 ------------------ ► 2NH3 + CO2
urea Ammonia Carbon dioxide
The ammonia reacts in solution to form ammonium carbonate, which results in an increase in the pH of the medium.
Urease activity is detected by inoculating a medium containing urea and a pH indicator, phenol red (yellow/beige/light
amber at acid pH and red-purple at alkaline pH). Initially the urea media chamber is mostly yellow. After incubation, a red-
purple color throughout the medium indicates a rapid urea splitter, a positive urease result. No color change, (the agar
remains yellow/beige/light amber) is a negative urease result. The urease test is included in the enterotube, see Image 1,
chamber 11. Escherichia coli and Enterobacter aerogenes does not normally have the enzyme urease. Proteus vulgaris is
a rapid urea splitter, as indicated by the bright purple chamber 11.
Decarboxylase tests are useful for differentiating bacteria. Microorganisms that have the enzyme decarboxylase
can remove the carboxyl group from an amino acid. Certain bacteria can decarboxylate the amino acid lysine using the
enzyme lysine decarboxylase, which results in the formation of the alkaline end product, cadaverine. Some bacteria can
decarboxylate ornithine (a product of arginine hydrolysis) using the enzyme ornithine decarboxylase (ODC), which results
in the formation of the alkaline end product putrescine. The presence of these decarboxylation byproducts, cadaverine and
putrescine, are indicated by a change in the color of bromcresol purple, the pH indicator in the enterotube medium, from
pale yellow (acidic) to purple (alkaline). The medium is covered with wax to provide anaerobic conditions. Any degree of
purple should be interpreted as a positive reaction. The medium remains yellow, a negative reaction, if decarboxylation of
lysine or ornithine does not occur. These tests are included in the enterotube
Image 3 Lysine and ornithine decarboxylation reactions. The enzymatic reaction catalyzed by ornithine
decarboxylase.The pyridoxal phosphate (PLP)-dependent ODC enzyme catalyzes decarboxylation of ornithine and
produces putrescine.
E. Citrate Utilization
This test detects those organisms which are capable of utilizing citrate, in the form of its sodium salt, as the sole
source of carbon. Organisms capable of utilizing citrate produce alkaline metabolites which change the color of the
indicator from green (acidic) to deep blue (alkaline). Any degree of blue should be considered positive. Certain
microorganisms will not always produce the ideal “strong” positive color change. Lighter shades of the same basic color
should be considered positive here. The citrate utilization is tested in the enterotube.
Watch Video 1: how to inoculate an enterotube performed in Microbiology labs at NC State. (5:08)
URL: https://youtu.be/3pmaDdZPLJg
Watch Video 2: how to interpret Enterotube results. Video by Professor B (5:23). URL: https://youtu.be/CwxvUq4lTZ4
Phenotypic methods
Throughout this semester, we have learned about many methods to characterize and identify bacteria. These methods
include characterizing cell shape (cellular morphology), identifying Gram status or specialized cellular features through
staining, growth requirements (oxygen, pH, temperature, etc), appearance of colonies (colony morphology), and through
biochemical reactions (enterotubes, selective and/or differential media types, etc.). All of these are
primarily Phenotypic methods of analysis and characterization, that is, the results are a product of the expression of their
genes.
Cellular morphology: cell shape--through microscopy
Staining characteristics: Gram status, cell structures such as flagella, endospores---microscopy
Growth characteristics: culturing requirements such as oxygen, osmotic pressure, temperature, colony morphology---
-culturing techniques
Biochemical characteristics: biochemical tests such as enterotubes, oxidase, catalase tests, selective/differential
media.
Genotypic methods
The last method used for the identification of bacteria is Genetic analysis through the use of nucleic acid probes or other
molecular techniques.
The application of molecular techniques for detecting and identifying pathogens is widely used. In particular, in
surveillance studies these methods provide reliable epidemiological data for tracing the source of human infections, such
as a foodborne illness outbreak. A wide range of molecular techniques (including pulsed field gel electrophoresis,
multilocus sequence typing, random amplified polymorphism deoxyribonucleic acid, repetitive extragenic palindromic,
deoxyribonucleic acid sequencing, multiplex polymerase chain reaction and many more) have been used for detecting,
speciating, typing, classifying and/or characterizing pathogens of great significance to humans.
The advent of the “molecular biology age” has provided a plethora of tools and techniques for the detection, identification,
characterization, and typing of bacteria for a range of clinical and research purposes. Previously, the identification and
characterization of bacterial species was largely done by phenotypic and biochemical methods (such as through
selective/differential media and biochemical tests that we have discussed in the last 2 modules), which relied on
preliminary isolation and culture.
While these methods continue to hold place in certain settings, molecular-based techniques have provided unprecedented
insights into bacterial identification and typing. To name a few examples, genotypic methods have enabled the
identification of a large diversity of previously unknown taxa, the characterization of uncultivable bacteria, and facilitated
metagenomics studies on large and diverse bacterial communities. Both clinical and research setting have provided in
depth insights into bacterial virulence, pathogenesis, antibiotic resistance, and epidemiological typing, as well as
Figure 1: Schematic PCR primers binding to the 16S rRNA gene for amplification from a bacterial chromosome.
In a PCR reaction, the is a series of steps that occur. Usually the dsDNA is denatured to ssDNA. At 55-58degC, a pair of
synthesized oligonucleotide primers anneal to the ssDNA that flank the sequence of interest. At 72degC, a thermostable
DNA polymerase will replicate the ssDNA to dsDNA sequences. The cycle repeats itself 20-40x to amplify the DNA.
Watch Video 1: how to set up a PCR reaction. Video by Hands-on DNA. (4:38) URL:https://youtu.be/95qOSslefMM
Image 1: Agarose gel electrophoresis of 16S rRNA PCR products. Note the 4 bands at the 1600 bp marker.
The 16S rRNA gene is frequently a target for those who are interested in identifying the genera or species of a bacterium.
If you amplify the entire gene (~1500 bp), that is more than sufficient for a sequencing results that will tell you the identity
of the bacterium.
Watch video 2: how to load and run an agarose gel. Video by Bio-Rad Explorer. (4:06)
URL: https://youtu.be/uAttNVEEEwY
Watch video 3: how to interpret a DNA gel. Video by Nicole Lantz. (2:30) URL: https://youtu.be/eDmaBtxym30
Procedures
Adapted from “GE Illustra PuRe Taq Ready to go PCR beads” guide
1. Obtain PCR bead tubes, which contain Taq polymerase (heat resistant enzyme) and other necessary reagents. Using a
sharpie, label the top of the tubes with PCR reaction number assigned in class. Make sure not to accidentally rub this
off when handling the tube and double check when you put the tube into the PCR machine that your labeling is still
visible.
2. Add 25 μL of Master mix (contains molecular grade water + 16S rRNA primers) into the PCR bead tube. The bead will
start to dissolve and slightly effervesce.
3. As you dispense the Master mix, insert the micropipette tip into the mix so that you actually see the small volume go
directly into the mix.
4. Using a micropipette tip, carefully touch the colony on the streak plate. A small, visible dab of cells that barely fill the
very end of the pipette tip will provide enough DNA template for the reaction.
5. Dip pipette tip into reaction mix and gently swirl for 5-10 seconds to dislodge cells. Cap the tubes. Avoid forming
bubbles.
6. Transfer tubes to thermal cycler.
7. Select appropriate program† to start cycling (about 2 hours).
8. Once cycling is complete, remove tubes and incubate on ice. Follow your instructor’s instructions about storage, and
follow up protocols to quality test the PCR products and prepare them for sequencing.
General guidelines
· Run gels stored at room temperature
· Keep samples uniform and load deionized water into empty wells
· Load gel within 15 mins of opening the pouch
· E-gel can only be used once
Procedure
Sample preparation and Loading gel:
PCR Clean-Up
The PCR clean-up process is performed using a commercial product. Depending on the availability of the different
commercial kits, your TA will determine and provide the kit to use in lab. Directions will be provided with the kit.
Phenotypic Analysis
Culturing Results
Explain any culturing results here. Did you use any selective and/or differential media types to determine characteristics
of your unknown?
Biochemical Tests
Explain any biochemical tests results here. Tests could include enterotube results, carbohydrate fermentation, casein
hydrolysis, oxidase, catalase, tests etc.
Does the phenotypic results align with your genotypic results?