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BIO 301 MICROBIOLOGY

MICROBIOLOGY
LABORATORY
MANUAL
Student Manual

Southern Leyte State University-Hinunangan Campus

MANUAL CONTENTS
INTRODUCTION TO MICROBIOLOGY LABORATORY

Welcome to the laboratory component of Microbiology! These laboratory sections have been designed to
enhance and bring to life the materials that will be covered in lecture by allowing direct observation,
experimentation, and application of techniques commonly used when studying the various microorganisms. The
student should expect this course to be challenging, informative, and hopefully enjoyable. The latter of the
expectations is achieved through preparation (read each exercise prior to your scheduled lab) and active
participation in the laboratory exercises.
For the sake of time (and to retain sanity) it is imperative that you prepare before class. Read the introduction
to the scheduled exercise, and familiarize yourself with the steps in each activity process (materials and
methods section). It is not expected that you understand everything, just be familiar with the activities.
You will be assigned a modified lab write-up as homework for each lab (the format is included in the appendix).
The introductory paragraph should include a purpose statement as well as an introduction to the material in the
background information. As you will soon realize, experimentation using microbes often requires incubation
time for growth in order to make a proper determination from your results. For most exercises results will not be
available until the following scheduled class. At that time a record should be made of your results in the given
area. Your results sections along with a conclusion paragraph will be assigned as a post-lab.

EXERCISES
Exercise 1 Introduction: Microscopy and Cell types
Exercise 2 Basic Procedure in the Microbiology Lab
Exercise 3 Aseptic Technique and Media Preparation
Exercise 4 Morphological examination: Differential Staining Techniques Introduction to
Biochemical/Metabolic Differentiation
Exercise 5 Culturing: Media Selection (Defined, Complex, Selective, & Differential) and
Inoculation techniques
Exercise 6 Media Selection and Metabolic Characterization Continued
Laboratory Practicum I - Basic Laboratory Techniques
Laboratory Practicum II - Identification of an Unknown Bacterium
Exercise 7 Quantification of Microorganisms Bacteria
Exercise 8 Control of Microorganisms Testing and Evaluation Techniques
Exercise 9 Transformation of E. coli
Exercise 10 Parasitology
APPENDIX
Possible Organisms Chart (for Identification of Unknown)

EXERCISE 1
INTRODUCTION: MICROSCOPY AND CELL TYPES
Introduction:
Microbiology is the study of very small organisms, microorganisms, which can only be viewed with
the aid of a microscope. There are several groups of organisms that fit into this category including
bacteria, cyanobacteria, fungi, and protists. Within this group there are several species interesting
to humans because of their ability to cause disease or their use in the food industry. Many of these
microorganisms are unicellular although some are multicellular. These organisms are extremely
diverse in cell type, size, color, and reproductive strategy.
When working with microorganisms one easy way to classify them is by their cell type. All cells
(including plant and animal cells) can be categorized as either prokaryotic or eukaryotic. The
primary difference between these two cell types is the presence of a membrane-bound nucleus. All
eukaryotic cells have a membrane-bound nucleus that houses the genetic material (DNA) in
addition to other membrane-bound organelles such as mitochondria and chloroplasts. Prokaryotic
cells lack a membrane-bound nucleus, their genetic material is located in a particular region of the
cell called the nucleoid. In addition to this difference, prokaryotic cells are much smaller than
eukaryotic cells. Examples of prokaryotic cells include bacteria and cyanobacteria (photosynthetic
prokaryotes). Eukaryotic microorganisms include fungi, protozoa, and algae.
In this lab, we will be getting some first-hand experience with the microorganisms described above.
Since the eukaryotic cells are larger than prokaryotic cells, this will be the best place to start. Once
you have a feel for these larger cells you are then ready to begin investigating the smaller
prokaryotic cells.

Fungi include unicellular and multicellular eukaryotic organisms. One thing all fungi share
is that they are non-motile heterotrophs that absorb dissolved organic material through
their cell walls and all but the yeasts metabolize aerobically. We will only be observing
yeast in lab. Yeasts are round unicellular microbes that are widely distributed.

Protists are for the most part unicellular, eukaryotic cells consisting of several groups. The
two groups of protists under investigation here are algae and protozoa. The primary
difference between these two groups is that algae are photosynthetic while protozoa are
described as animal-like because they are heterotrophs (consume other protists, bacteria
and detritus). There are several protozoa that cause disease including Plasmodium vivax
(malaria) and Giardia lamblia (gastroenteritis).

Prokaryotes will be the primary focus of the semester. In todays lab we will observe
some of the diversity of this group by looking at both bacterial cells and cyanobacteria.
Remember that these cells are much smaller than eukaryotic cells, and lack membranebound nuclei.

In order to investigate microorganisms we need to become intimate with our primary tool--the
microscope. This invaluable tool allows the viewing of objects/structures that otherwise would go
unnoticed by the unaided human eye.
The type of microscope shown below is called the light microscope. Light is conducted through
curved lenses in such a way that an object may be viewed larger than its actual size. You might
want to label the basic structures and take notes as your instructor goes over the microscopes
structure and function.

objective
and 100X.
is the
by the
instance when
lens, the

The light microscopes used in this lab are

binocular and have ocular lenses with a
magnification of 10X. In addition to this
magnification there are also four different
lenses to choose from 4X, 10X, 40X,
Magnification of the object being viewed
product of the ocular objective multiplied
lens objective currently in use. For
viewing an object on the 4X objective
object is magnified a total of 40X.
Even the highest quality light microscope is limited
in its magnification abilities. The highest objective
for our microscopes is 100X, which has a
magnification of 1000X. With this magnification
even the slightest distortion of light would greatly
reduce the quality of the image. In order to
conduct the light properly at this objective,

it is necessary to place a drop of oil on

the slide and immerse the lens. The oil
immersion lens (100X) is specially sealed and is

it is

top of

the only lens that should be placed in oil. We will learn more about the oil immersion lens
in E2.
The importance of proper handling and use of the microscope is vital. You will find this to be
especially true as beginning microscopists. It is critical that you clean the microscopes before and
after use. Please take notes while your instructor goes over this information with you.
Materials Needed:
Blank
Slides/Cover
slips Iodine
10%
bleach
soln. Cultures:
Yeast
Yogurt
Gleocapsa

Oscillatoria
Anabaena
Volvox
Spirogyra
Amoeba
Paramecium

Activities
In order to observe the differences between these cell types and become familiar with the
microscope you will be preparing several slides primarily using the wet mount technique
as described below. In some cases prepared slides may be provided.

Remember to CLEAN your microscope before starting to view your slides.

Use a
new KimWipe to gently wipe the ocular lenses and then wipe the 10x, 40x, and 100x objective
lenses. If there is any excess oil on the microscope be sure and remove that. If you have
trouble removing oil, use the microscope cleaner provided.
For each activity below, use the wet mount method to make your slides. Add the drop of iodine as
described for those cultures with an (*).
1. Add a drop of live culture
2. Add a cover slip and then add a drop of iodine* beside the coverslip.
3. Observe under the microscope up to 40x and sketch your results in table provided
4. Place slide in 10% bleach solution provided

5.
6.
Activity
I:
Fungus
Yeast
Observation*
7.

8.

9.

(http://bugs.bio.usyd.edu.au/learning/resources/CAL/Microconcepts/images/Topics/Diversity/buddingYeastCells.jpg

10. Activity II: Protists

A) Protozoa
11. Amoeba *

27.

13.
14.
15.
16.
17.
18.
19.

http://www.hinsdale86.or
g/staff/kg
abric/DIMACS/amoeba.jpg

29.
30.
B) Green Algae

31. Volvox
33.

28.

12. Paramecium *
20.
21.
22.
23.
24.
25.
26.

http://www.educa.madrid.
org/web/i
es.alonsoquijano.alcala/carpetas/q
uienes/departamentos/ccnn/web_
1_ciclo_ESO/1eso/images/tema10/paramecium2.jpg

32. Spirogyra

34.

36.

37. 35.

http://www.lima.ohiohttps://www.msu.edu/course/bot/423/
state.edu/biology/images/volvo
Spirogyr a.jpg
x.jpg

38.

39. Activity III: Prokaryotes

A) Cyanobacteria
40. Gleocapsa

43.

64.

http://www.glerl.noaa.gov/seagrant/
GLW L/Al
gae/Cyanophyta/Images/Gloeocapsa2.jpg

Southern Leyte State University-Hinunangan Campus

65.

41. Anabaena
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.

http://www.bo
m.hik.se/n
esch/kac/anabaena.jpg

66.

42. Oscillatoria
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.

http://silicasecchidisk.conncoll.edu/Pics/Ot
her%20
Algae/Blue_Green
%20jpegs/Oscillatoria4.jpg

67.

B) Bacteria
C) Lactobacillus (yogurt) *
No photo available

Southern Leyte State University-Hinunangan Campus

68.
69. Remember to CLEAN your microscope before putting it in the cabinet.
70.
71. E1 RESULTS:

72.
73. Eukaryotic Cells
75.
74.
Type
76.
Sketch on (40x)
77.
79.
78. Fungi:
Yeast

80.
81. Protist:
83.

82.

Amoeba

84. Protist:
85. Parameciu
m

87.
88. Protist:

86.
89.

Volvox

90.
91. Protist:

92.

Spirogyra

93.
94. Prokaryotic Cells
95.
97.
96.
Type
98.
Sketch (40x)
99.
101.
100.
Cyanobacteria: Gleocapsa
102.
104.
103.
Cyanobacteria: Oscillatoria
105.
107.
106.
Cyanobacteria: Anabaena
108.
110.
109.
Bacteria: Lactobacillus (yogurt)
111.
112.
E1 Write-up
113.
Introduction paragraph: include a description of the following items from the
background information:
Prokaryotic vs. eukaryotic cells
Introduction of types cells viewed
114.
Submit results from lab manual
115.
Conclusion:
Draw conclusions about the importance of getting familiar with proper
microscope technique (for instance proper handling, focusing, and cleaning of
the microscope).
Include a comparison of the prokaryotic and eukaryotic cells observed in this class.

116.
117.

This write-up must be typed and be in your own words.

Southern Leyte State University-Hinunangan Campus

119.

118. EXERCISE 2
BASIC PROCEDURE IN THE MICROBIOLOGY
LABORATORYIntroduction

120.
121.
Microbes can serve as either our greatest allies or our worst enemies depending on their
type and location. This is why studying these organisms is so vital. Methods for studying microbes
are as diverse as the groups themselves.

122.

123.
Due to their size and ubiquity, microbes can be a challenge in the laboratory. This unit will
focus on the general techniques that are needed in order to achieve good experimental outcomes
while protecting the health and safety of everyone. We will be focusing on two important tools in the
microbiologists tool boxworking with bacteria cultures and microscopy.

124.

125.
T
he first part of the laboratory will introduce you to the materials and methods we routinely use when
working with bacterial cultures. The most important technique to learn today is aseptic technique
which will be explored in greater detail in E3. In addition to learning this basic technique we will also
focus on a key idea, that our world is full of bacteria present virtually everywhere. Due to the
ubiquity of microorganisms we will have to be extremely careful about contamination of our lab
materials and the cultures we are working with. You will see that it takes very little exposure to
introduce an unwanted organism to your materials.

126.

127.
The second part of the laboratory will continue our microscopic investigation into the
microbial world by introducing you to the variety of morphologies (shapes) and cellular arrangements
that are present among bacterial cells as well as comparing cell size. In addition, we will also focus
on using the oil immersion lens which is a critical skill in the microbiology lab.

128.
129.
Cellular morphology and arrangement refers to the cell shape and the association shared
between cells (if any). Although bacterial cellular morphology can be very diverse, there are three
basic shapes of interest in our lab. They are coccus (pl. cocci), rod (sometimes called bacillus), and
spiral. Based on how cocci cells divide they can designated as diplococci (pairs), streptococci
(chains), tetrads (groups of 4), or staphylococci (clusters). Rod cellular arrangements can be
described as singles, diplobaccilli (pairs), or streptobacilli (chains).

130.
131.
When comparing bacterial cell size, we will be using the metric system. The metric system is
the standard system of measurement used in the sciences, including microbiology. The system
makes measurement and unit conversions much simpler because all units and conversions are
based upon the number 10.

132.

133.
Three of the most commonly measured properties are length, mass, and volume. The
standard metric units for these variables are meter, gram, and liter. Prefixes are placed in front of
each units name to designate smaller and larger units. Lets use length as an example to see how
metric units work. One meter is equivalent to 39.37 inches, so it is roughly 1 yard in length. Now
imagine dividing the meter into 10 equal-sized pieces. Each piece is 1 decimeter (dm) in length
th
134.
(1/10 of a meter). Imagine dividing the meter into 100 equal-sized pieces. Each piece is 1
th
135.
centimeter (cm) in length (1/100 of a meter). Finally, divide the meter into 1000 pieces.
th
Each piece is 1 millimeter (mm) in length (1/1000 of a meter).

Southern Leyte State University-Hinunangan Campus

Bio 301 MICROBIOLOGY

136.
137.
139.

138.

Note the following:

140.
1 m = 10 dm
141.
1 m = 100 cm

142.
143.

1 m = 1000 mm
1m = 1,000,000 m

144.
145.
called
in

146.

Most microorganisms are smaller than 1 mm, so we need to introduce an additional unit
the micrometer (m). Imagine dividing 1 mm into 1000 equal-sized pieces. Each of these
extremely small pieces is 1 micrometer in length. A typical bacteria cell is about 0.2-2.0 m
diameter and 2-8 m in length.

147.
Morphology/Arr
150.angement
151.
152.
Rods Singles

148.
Sp
153.ecies
154.
155.
Esc

149.

Photo

156.

herichia coli

157.
158.
159.

streptobacilli

164.
165.
166.

Tetrads

171.
172.
173.

staphylococci

177.

163.

Bac
illus subtilis

167.
168.
169. Microco

170.

ccus luteus

174.
175. Staphyloc

176.

occus aureus

183.
184.
185.
186.

178.
Materials per pair:

179.
180.
TSA plates
181.
sterile swabs
182.
187.
188.

160.
161.
162.

sterile water
prepared bacterial slides

Activity I: Ubiquity of M icroorganisms

189.
The specific media type used in this lab is Trypticase Soy Agar (TSA) which is a
complex media
190.
providing a wide range of nutrients supporting a diversity of microorganisms.

191.
192.
193.
194.
195.
o

196.37
C

202.
203.
204.2
5

197.

Ubiquity of Microorganisms: Swab Samples

1. Obtain 3 TSA plates, flip the plates over, and label the
bottom with your group initials, lab day/time.
2. Label one plate air. Remove the lid and leave it exposed
to the air until the end of the lab period.
3. Add the following information on the remaining 2 plates:
a. Divide each plate into quadrants

b. Label one plate 37 C and the other 25 C.

198.

199.
200.
inition of
Def
Fomite: nanimate
anobject
i can transfer
that
201.
ase i.e.
dise
door b
kno

Bio 301 MICROBIOLOGY

c. N
u
m
b
e
r
e

206.

ach section 1-4, and make a key below of the four samples you would like to
swab. You must use the same four sample areas for both plates
205.
and these should include one fomite sample, two body samples, and
one surface sample

207. Source 1: dirty finger

3:__________________
208. Source 2: clean finger
_________________

Source
Source 4:

BIOL1230 MICROBIOLOGY

209.
210.
211.
212.

4. In order to inoculate sources 1 and 2 start with dirty hands. Moisten your index
finger with water then press your dirty index finger into quadrant 1 of both plates.
5. Now wash your hands following these directions from the Center for Disease Control:

213.
a.
b.
c.
d.
e.

Wet your hands with warm running water and apply soap.
Rub hands together to make lather and scrub all surfaces.
Continue rubbing hands for 20 seconds. (Imagine singing "Happy Birthday" twice)
Rinse hands well under running water
Dry your hands using a paper towel and use your paper towel to turn off the faucet.

214.
215.
216.

217.

218.

6. Moisten your index finger again and press the clean index finger into quadrant 2
of both plates.
7. For sources 3 and 4: obtain two sterile swabs and one micro vial of water from
the cart.
8. For source 3, moisten one swab and streak the area of interest thoroughly.
Proceed to streak each of your TSA plates in the designated quadrant for that
sample. Be suretorememberaseptictechnique! (repeat for source 4)

9. Place the 37 C plate upside down (bottoms up) inside the incubator. Place the 25
C plate upside down on top of the incubator. Since microbes are so small, it is
necessary to allow time (24-48 hours) for them to multiply into populations so large
we can see their colonies unaided. The plate incubated at room temperature will
need a longer incubation (5-7 days).

219.
We will be discussing throughout the semester basic requirements microbes have in order
to live and replicate. These requirements include nutrition and environmental conditions such as
temperature, pH, moisture, and oxygen. Microbes vary in their nutritional needs and their levels of
tolerance to environmental conditions. Since both plates were streaked with the same samples
and supplied the same nutritional media, it is interesting to expose each plate to a different
o
o
220.
variable in this case temperature (either 25 C or 37 C). Both plates will be
incubated in aerobic atmospheric conditions.

221.
222.
Activity II: Cellular Morphology and Arrangement (prepared
slides)
223.
The purpose of this second activity is 2-fold:
1) introduction to cellular morphology and arrangement
2) proper use of the oil immersion (100x) objective lens.

224.

1. Obtain 3 prepared slides representing different morphologies and arrangements.

2. Begin focusing each slide on the 4x and then move to the 10x and 40x objective
lenses. Once you have your image focused clearly on 40x move the turret in
between the 40x and 100x objectives.
3. Without moving the stage or changing focus, drop a small, single drop of
immersion oil onto the slide.
4. Move the oil immersion (100x) objective lens directly into the spot of oil.
5. View slideyou might have to do some minimal focusing with the fine
adjustment knob to clear up your image.
6. Sketch results in table provided.
7. Clean oil off 100x object lens before moving to the next slide.

AIR PLATE

225.
you should have left this plate uncovered on
your lab bench the entire lab. At the end of the period be sure to close your dish

BIOL1230 MICROBIOLOGY

and invert and incubate at 37 C.

226.

227.

Clean-up and Disposal follow instructors directions.

228.
Once all items have been put in their assigned places wipe
your work area with ethyl alcohol and wash your hands well.

229.
230.

E2 Results:

231.
ACTIVITY I
RESULTS: Obtain
your two sample plates
you swabbed last
232.
lab session
and look for growth.
Use the colony
description figure
taken
233.
from Science Buddies website
234.
( http://
www.sciencebudd
ies.org/me
ntoring/project_ide
as/MicroBio_img_
003.gif) to assist
you in describing
bacterial growth
for each
quadrant. The
results tables
provided below
are to be used to
briefly describe
what you
observe. List
each sample area
in the space
provided. The
lower portion is
for growth
description.
Include color,
colony sizes,
amount of growth,
and colony
description for
each listed
sample area.

235.
236.

237.

Colony Description by Source for 37 C Plate

238.

Source 1:
dirty index finger

243.

Amount

244.

escripti
on

239.
D

Source 2: clean
index
240. finger

245.

Amount

246.

escripti
on

241.

Source 3:

247.

248.

Amount

escripti
on

242.

249.

Source 4:

Amount

250.

Descr
ip

BIOL1230 MICROBIOLOGY

251.

252.

253.

254.

255.

256.

257.

258.

259.

260.

Colony Description by Source for 25 C Plate

261.

Source 262.
Source 2: clean index
1: dirty index
263.
finger
finger
266. 267.
D
268.
269.
D
Am
escriptio
Amount
escriptio
n
n

274.

282.
283.
284.
285.
286.
287.

275.

276.

277.

264.

Source 3:

265.

270.

271.

272.

278.

279.

280.

Amount

D
escriptio
n

Source 4:

Amount

273.

Des
c
281.r

AIR Plate Results: Did you have growth?

288.
ACTIVITY II RESULTS: For each slide sketch enough cells to demonstrate
the morphology and
289.
arrangement of the bacteria viewed using the oil immersion (100x)
objective lens.

290.
291.

292.

Slide 1

293.

Slide 2

294.

295.

298.

299.

300.

304.

305.

306.

296.

S
KETCH

297.

301.
De
scribe
302.
m
orpholog
y and
303.
ar
rangeme
nt

307.

308. E2 Write-up
309. Introduction paragraph: include a description of the
following items from the background
310. information:
311. Ubiquity of microorganisms and aseptic technique
312. Discussion of culturing conditions provided including
temperatures, media used, and
313. atmospheric requirements

Slide 3

BIOL1230 MICROBIOLOGY

314. Description of different morphologies and

arrangements
315. Submit results from lab manual
316. Conclusion:
317. Use your data to support use of aseptic technique
318. Use your data to address the use of optimal
temperature during incubation

319.

Discuss your observations of cellular morphology and

arrangement
320.

321.

This write-up must be typed and be in your own

words.
322.
323.
324.
325.
326.
327.
328.
329.
330.
331.
332.
333.
334.
335.
336.
338.

337.
EXERCISE 3
ASEPTIC TECHNIQUE AND MEDIA PREPARATION

339.
340.
341.

342.

Introduction

343.
344.
Asepsis means without contamination. The ability to carry
out procedures without the
345. introduction of unwanted organisms, or contamination, is
paramount to obtaining correct
346. results/identification. In addition, since some of these
microbes are potential pathogens
347. (organisms that cause disease), contamination could expose
you and any other person you may come into contact with the
possibility of infection. That is why vigilance in proper technique
is necessary to reduce risk.

BIOL1230 MICROBIOLOGY

348. Aseptic technique can be further divided into four categories

including work area preparation,
349. media preparation/handling, culture transfer, and clean-up
and disposal.
350. Work.
351.

352.
354.

Work Area Preparation

353. Since microbes are everywhere, even on us, it is necessary
to begin to minimize the potential for contamination before we
begin to work with the microbes. Prior to beginning each
laboratory session.

355.
356.
357.
358.

359.
Results: Did you have growth?

AIR Plate

360.

361. ACTIVITY II RESULTS: For each slide sketch enough cells to

demonstrate the morphology and arrangement of the bacteria viewed
using the oil immersion (100x) objective lens.
362.

363.

assigned microscope #:

364.

366.

365.
369.

370. SKE
TCH
374. Describ
378.
379.

Sli

367.

Sli

368.

371.

372.

373.

375.

376.

377.

Sli

380.
E2 Write-up
381.
Introduction paragraph: include a description of the following
items from the background information:
Ubiquity of microorganisms and aseptic technique
Discussion of culturing conditions provided including
temperatures, media used, and atmospheric requirements
Description of different morphologies and arrangements
382.
Submit results from lab manual
383.
Conclusion:
Use your data to support use of aseptic technique
Use your data to address the use of optimal temperature during
incubation
Discuss your observations of cellular morphology and arrangement.
384.

385.
386.
387.
388.
389.
390.
391.

This write-up must be typed and be in your own words.

392.
393.
394.
396.

395.
EXERCISE 3
ASEPTIC TECHNIQUE AND MEDIA PREPARATION
397.
398.
399.

400.
Introduction
401.
Asepsis means without contamination. The ability to carry out procedures
without the
402.
introduction of unwanted organisms, or contamination, is paramount to
obtaining correct
403.
results/identification. In addition, since some of these microbes are potential
pathogens
404.
(organisms that cause disease), contamination could expose you and any
other person you may
405.
come into contact with the possibility of infection. That is why vigilance in
proper technique is
406.
necessary to reduce risk.
407.
Aseptic technique can be further divided into four categories including work
area preparation,
408.
media preparation/handling, culture transfer, and clean-up and disposal.

409.
410.
Work Area Preparation
411.
Since microbes are everywhere, even on us, it is necessary to begin to
minimize the potential for
412.
contamination before we begin to work with the microbes. Prior to
beginning each laboratory
413.
session:

414.
415.
Remove all you personal items away from the workbench except for your lab
416.
notebook and writing instrument.
417.
Wipe down your workbench with ethyl alcohol (ETOH) and paper towels.
418.
Wash your hands using CDC method as described in E2.
419.
Begin gathering materials as instructed and outlined for that particular
laboratory.

420.
421.
Media Preparation/Handling
422.
When culturing bacteria you must provide them with all of the conditions they
need for growth
423.
including nutrients, temperature, atmospheric requirements and more. Media
is the nutrient
424.
mixture that is used to grow and keep microbes. Media is usually in broth or
solid forms and will
425.
vary in content based on the goal of its use. These nutrient mixtures are
normally in powdered
426.
form to increase their shelf life. When needed, the nutrient mix is added in a
specific amount to
427.
water, boiled, transferred to containers, and autoclaved to sterilize. As long as
the media remains
428.
unopened in the original container, it should remain a sterile environment. If
the media is

429.
transferred from the original container, care should be taken to avoid
contamination. In the case
430.
of the plastic Petri plates we will be using extensively in this lab section,
media must be
431.
transferred. Plastic used for these plates are unable to withstand the
temperature of the autoclave
432.
for sterilization so they are shipped and remain in sterile packaging until their
time of use. The
433.
agar will be poured hot and quickly into the Petri dish to further avoid
contamination.

434.

435.
Culture Transfer
436.
All of microbiology work involves transferring cultures to different growth
media or onto slides. It
437.
is critical when transferring bacteria that aseptic technique is maintained. A
large portion of
438.
todays lab procedure will emphasize how to move bacteria to different
media types while
439.
preventing contamination.

440.
441.
Clean-up and Disposal After EACH Lab (last step of aseptic
technique)
442.
Unless otherwise instructed items used in the lab session should be
treated in the following manner:
443.
Glassware - will be autoclaved and cleaned for re-use.
Test tubes/flasks place in the designated test tube rack on the lab cart after the
444.
labeling has been removed. If an adhesive label is present, simply
peel it off and
445.
place in the autoclave bag. If a marker has been used, wipe off
with acetone prior to
Slides - If the slide has been heat fixed, wash with sponge and dish soap and place
in designated container.

446.

447.
Disposable Items - items intended for one time use and items that cannot
maintain their form
448.
and function after being autoclaved should be placed in the autoclave bag.
These items will be
449.
sterilized prior to their disposal to avoid contamination during and after waste
collection. These
450.
items include but are not limited to Petri plates and any paper or plastic
items that have come into contact with microbes.
452.

451.
Goggles clean with diluted dish soap, dry and then put away.

453.
454.
Once all items have been put in their assigned places wipe your
work area with ETOH and wash your hands.

455.
456.
457.
Activities
458.
Materials:
459.
4 test tubes of TSB
460. 1 TSA plate
461.
plates from E2

hot pads
flasks with filtered water
powdered media

462.
463.

stirrer/hotplate
magnetic stirrer

thermometers

464.
465.
466.
467.

468.
469.

Aseptic Transfers
A.) Sterile Broth to Sterile Broth:

A.) Sterile Broth to Sterile Broth:

1. Obtain two sterile Trypticase Soy Broth (TSB) test tubes.

470.
471.

Label one tube A and one tube B and label both with your group initials and lab
day/time.

472.

2. Flame loop until the loop and some of the wire is red hot then let the loop cool.

473.
474.
3. Open test tube A using the pinky finger techniquehold the lid of the test tube with your
pinky finger of your dominant hand, and use your other hand to twist the test tube away from
the lid.

475.

4. Flame the lip of the test tube

476.
477.
5. Get loopful of broth; make sure that only the sterilized portion of the loop makes contact
with the broth.

478.

6. Flame and close test tube.

479.

7. Open test tube labeled B using the same technique as above.

480.

8. Flame the lip of the test tube.

481.

9. Inoculate test tube B with the loopful of sterile broth from tube A. Be sure that only the
sterilized portion of the loop makes contact with the broth.

482.

10. Flame and close test tube B.

483.
484.
485.
486.
487.
488.
489.

11. Flame loop red hot.

12. Place both test tubes in a rack in the incubator.
B.) Sterile Broth to Sterile Plate:

B.) Sterile Broth to Sterile Plate:

1. Obtain one sterile TSB test tube and a sterile Trypticase Soy Agar plate (TSA). Label both with
your group initials and lab day/time. Label the test tube C and the plate D. Be sure to put your
labels on the bottom of the Petri dish.

490.

2. Flame loop until the loop and some of the wire is red hot then let the loop cool.

491.

3. Open the test tube using the pinky finger technique (see above)

492.
493.
4. Flame the lip of the test tube

494.
5. Get loopful of broth; make sure that only the sterilized portion of the loop
makes contact with the broth.

495.
496.

6. Flame and close test tube.

497.

7. Open the Petri dish just enough to get the loop in.
8. Very gently move the loop across the Petri dish. Be careful not to dig into or make any
breaks in the agar bottom.

498.

9. Close the Petri dish.

499.

10. Flame loop red hot.

500.

11. Invert the Petri dish and add it to your class stack in the incubator. Put the test tube in the
incubator rack too.

501.

502.

503.
504.
505.
506.

C.) Colony to Broth Transfer

1. Obtain one sterile TSB test tube and a plate with growth from E2. Label with your group
initials, lab day/time and E on the TSB tube.

507.

2. Flame loop until the loop and some of the wire is red hot. Allow loop to cool.

508.

3. Open the Petri dish just enough to get the loop in and very gently use your loop to pick up an
isolated colony of bacteria. Be sure not to disturb the agar or any neighboring
509.
colonies.

510.
511.
512.

513.

4. Close the Petri dish.

514.

5. Open the test tube using the pinky finger technique (see above).

515.

6. Flame the lip of the test tube

516.
517.
7. Inoculate the broth; make sure that only the sterilized portion of the loop makes contact with the
broth.

518.

8. Flame and close test tube.

519.

9. Flame loop red hot.

520.

10. Put the test tube in the incubator rack.

521.
522.

523.
524.
525.
526.
527.
528.
529.
530.
531.
532.
533.
534.
535.
536.
537.
538.
539.
540.
541.
542.
543.
544.
545.
546.
547.
548.
549.

550.
551.
552.
553.
554.
555.
556.

Media Preparation
557.
Each group will be asked to prepare media. The type of media and the specific
instructions will be
558.
assigned by the instructor. It will take just a few minutes to get the media
measured and added to
559.
the flask. It will take about 20-30 minutes for the media to reach boiling before
it is ready to pour
560.
into test tubes. Instructors will need to make sure that each class tubes are
autoclaved as soon as possible and then stored in the refrigerator as room allows.
The basic procedure to be used
561. by all classes is as follows:

562.
563.
1. Measure out the proper amount of media powder for L of media using the
electronic
564.
balance.

565.
566.
2. Fill up your flask with the proper volume of filtered water (1/2 L) and place
on the
567.
stirrer/hotplate. Drop in a magnetic stirrer and turn the stirrer on 6-8.

568.
569.

3. Slowly pour in the media powder into the water.

570.

571.
4. Turn the hotplate on high. Agar can superheat so it is important to keep an
eye on your
572.
boiling agar. Also agar will not dissolve in water that is hot, so DONT heat the
water until
573.
after adding the powder.

574.
575.
5. Once the agar or broth has reached boiling point (use thermometers to
register 100oC). You
576.
will notice that the liquid is clear rather than cloudy indicating that the media
has dissolved.
577.
Also there is often a frothy head that forms on the top of the boiling media
(when TSA and
578.
TSB start to look like beerthey are ready).

579.
580.
6. Follow instructors directions on how to pour the media into the test tubes
given. Loosely cap
581.
the test tubes and put autoclave indicator tape on the rack. Be sure to provide
a label on the
582. rack with the type of media, date made, and group initials. Your instructor will help
you get

583. your rack autoclaved with the rest of the class tubes.

584.
585.
7. Test tubes need to be autoclaved as soon as possible after being poured.
Once sterilized
586. test tubes can be stored in the refrigerator.

587.
588. E3 Results
589.
For each item record whether or not you had growth. If you have growth in
TSB then the test
590. tube will be cloudy. You might need to vortex your TSB to see growth as the bacteria
can settle.

591.
592.
593.
594.
599.
600.

Procedure
Sterile TSB -7

Sterile TSB

608.
609.

Sterile TSB -7

Sterile TSA

617.
618. Isolated colony -7 sterile

595.
596. Materials
601.
602.
605. Tube A
606.
610. Tube B
611. Tube C
614.
615. TSA plate D
619.
620.

TSB

597.
598.

Growth? Yes or

No

603.
607.
612.
616.
621.

Tube E

622.
623.
624.
625.
626.
E3 Write-up
627. Introduction paragraph: include a description of the following items from the
background information:
1. Aseptic technique define, state purpose, give some examples of
628.
Submit results from lab manual
629.
Conclusion:
2. Did your results turn out as expected? Explain.
3. Describe some procedural errors that could negatively affect your results.

630.
631.
632.
633.

This write-up must be typed and be in your own words.

.

634.

EXERCISE 4

635. DIFFERENTIAL STAINING TECHNIQUES GRAM STAIN AND SPECIAL

636. STRUCTURE STAINS

637.
638.

Introduction

639. As we have seen in the previous exercises, light microscopy and the use of a stain are
valuable tools for viewing bacteria. This allows us to see the morphology of a microbe of interest. A
differential stain technique allows additional discrimination and helps to narrow down the list of
possibilities with regard to its identity. A differential stain would be one that allows determination
640.
of differences between species having similar morphologies based on their ability to take
the stain.

641.

642.In multiple stain procedures, the initial stain is retained by cells that are termed positive while the
negativecell loose the initial stain. This necessitates the addition of a secondary or counter stain, to
make the negative cells visible under the microscope. Differential stains show a difference in
chemical composition, metabolism, and/or the presence of a special structure.

643.
644.
The first step in making a slide is to smear the bacteria onto the slide.
Preparation procedures for
645.
the smear will vary according to cell concentration in the culture. Generally a
broth culture will be
646.
less concentrated than surface cultures and can be smeared directly onto the
surface of a clean
647.
slide and allowed to air dry. Surface cultures (ones growing on agar) need to
be diluted in order to
648. discriminate single cell shapes once stained. A loop of water is added to the slide and
the loop of sample is mixed in and distributed on the surface of the slide and allowed to dry.

649.
650.
The second step in making most differential stains is to heat fix the slide. A
heat fixed slide is
651.
one where the organisms are placed on the slide and the slide is allowed to
dry. After the slide is
652.
free of visible moisture, it is passed through an open flame 2-3 times before
the staining begins. It
653.
is imperative that the slide is completely dry before the heat-fix step;
otherwise the bacterial
654.
proteins can be denatured during the heat-fix step and cause a distortion in
the cell morphology.
655.
The purpose of the heat-fix process is to affix them to the surface of the slide
so that they are not
656.
washed off during the staining process. When done correctly, heat-fixing will
also kill the
657.
organisms.

658.
659.
The most well-known and used differential stain is the Gram stain. This stain
process
660.
determines differences in peptidoglycan (a chemical found only in bacteria) on
the outside of the
661. cell wall. Bacteria can be divided into one of two categories based on their gram
reaction, either gram positive or gram negative. Gram positive cells have a thicker
peptidoglycan cell wall than gram-negative cells, and retain the initial stain, crystal violet
(purple), in the gram stain procedure. Gram negative cells are decolorized during the gram
stain procedure which requires the application of a counter stain, Safranin (pink), so that
these cells can be easily viewed.

662.

663.
664.
The acid-fast stain is used in a limited number of cases to stain organisms
that have an addition
665.
of mycolic acid in their cell walls which prevents them from Gram staining.
The Mycobacterium
666.
species require this type of stain process in order to view them. Due to the
nature of the waxy
667. mycolic acid, these cells must first be steamed in order for the stain to penetrate the
cell wall.
668.
In this procedure acid-fast cells retain the initial stain, carbolfuchsin (reddishpurple), while negative
669.
Cells are stained by the secondary stain, Methylene blue (blue).

670.
671.
Some species have cellular structures that are uncommon and can be used in
identification.
672.
Endospores are examples of specialized cellular structures produced by some
Bacillus and
673.
Clostridium species in response to stressful environmental conditions.
Endospores are dormant
674.
cells able to survive hazardous conditions that later germinate to form
vegetative (metabolically
675.
active) cells when conditions improve. The endospore stain employs a
multiple stain
676.
procedure that stains both endospores and vegetative cells. The endospore
stain involves steam
677.
similar to the acid-fast stain. Without steam the tough spore coat prevents the
endospore from
678.
taking up stain. During this procedure, endospores keep the initial stain,
Malachite green (green),
679.
while the vegetative cells retain the secondary stain, Safranin (pink).

680.
681.
Other important structures that can be detected with stains are flagella. These
structures are
682.
Responsible for the motility of an organism. While the presence of flagella
alone can be important
683.
in characterizing bacteria, the ability to describe the arrangement of the
flagella can be even more
684.
useful. For direct observation of flagella a tricky staining procedure is used.
Motility media is an
685.
alternative method to staining that allows for indirect observation of flagella.
Due to the dilute
686.
nature of this media microbes are able to travel in the media if they are
mobile. This takes time
687.
And incubation will be necessary. In addition the media has a chemical added,
TCC, which
688.
microbes take into their cells. When metabolized, the TCC changes from being
colorless to a pink
689.
color allowing easier viewing of the placement of the growth.

690.
691.
There are plenty of resources on the internet that can help you visualize these
processes a little better. Check out the following links:

692.
693.

Gram Stain: http://faculty.mc3.edu/jearl/ML/mL-5.htm

694.
Acid-fast Stain: http://faculty.mc3.edu/jearl/ML/mL-6.htm
695.
Endospore stain:
http://www.slic2.wsu.edu:82/hurlbert/micro101/pages/101lab6.htmL

696.
697.

698.

699.
Activity
700.
Materials:
701.
Per Class:
702.
Blank slides
703.
Hotplates/beakers
media test tubes

Per team
M. smegmatis (pathogenic)
B. subtilis
E. coli

K. pneumonia
S. epidermidis
P. aeruginosa
3 motility

704.
705.
706.

Students will have 2 lab periods to complete E4.

707.

708.
Each group should start by inoculating the 3 motility tubes
709.
1. Obtain 3 test tubes of motility media and label 1-3, group initials, and
class period.
710.
711.
712.

1. Escherichia coli
2. Pseudomonas aeruginosa
3. Klebsiella pneumoniae

713.
2. Using aseptic technique, stick the sterile inoculation needle into the broth
culture (be sure to flame the
entire length of the needle).
714.
3. Insert the needle straight down into the motility media and pull the needle
straight back out.
715.
4. Fire the tube and quickly replace the lid. Fire the needle
716.
5. Place in the test tube rack in the incubator for 48 hours.

717.

718.
Each student should obtain a total of 8 slides and label slides
a-h (see
719.
List of bacteria below). Make all 8 slides using the heat-fix
procedure. After
720.
Heat-fixing all 8 slides begin staining slides starting with the
4 gram stain slides.

721.
722.
Sli
de label
725.
a
and f
728.
b

731.
c
and h
734.
d
and g
737.
e

740.
i

723.

Organism

724.
Stain
Technique

726.
Staphylococcus
epidermidis
729.
Pseudomonas aeruginosa

727.
Gram stain
and acid-fast stain
730.
Gram stain

732.

Escherichia coli

735.

Bacillus subtilis

733.
Gram stain
and endospore stain
736.
Gram stain
and endospore stain
739.
Acid-fast stain

738.
Mycobacterium smegmatis
(PATHOGENIC)
741.
MIXED slide of S.
epidermidis and E. coli

742.

Gram stain

743.
744.
745.
746.
747.

For All slidesmake smear using heat-fix procedure:

748.
From Solid Sample:
Sample:

749.

1. Clean and label bottom of slide

From Liquid
1. Clean and label

bottom of slide

750.
751.
752.
753.

2. Add loopful of water

3. Flame loop red hot
4. Aseptically obtain bacteria (isolated
colony if possible)

2. Flame loop red hot

3. Vortex Sample
4. Flame test tube opening
5. Aseptically obtain

bacteria

754.

5. Smear (spread-out) bacteria on

6. Flame test tube

opening

755.

Slide

7. Smear (spread-out)

bacteria on slide

756.

6. Flame loop red hot

757.

7. AIR DRY COMPLETELY

8. Flame loop red hot

9. AIR DRY

COMPLETELY
758.

8. Heat-fix (pass slide through flame)

10. Heat-fix (pass

slide through flame)

759.

9. Proceed to directions below for the

below for the

760.
specific differential stain.
stain.

11. Proceed to directions

specific differential

761.
762.
763.
Under oil immersion, observe your slides--remember you are looking for
differences in
764.
morphology, size and arrangement in addition to whether the organism is
positive or negative for
765.
the differential stain. Then sketch your observation in the results section.
(Dont forget to clean
766.
your microscope BEFORE & AFTER use.)

767.
768.
769.
770.
771.
772.
773.
774.
775.
776.
777.
778.
779.

780.
781.
782.
783.
784.
785.
786.

Procedure I

788.
789.
790.
791.
792.
793.

Organisms:
a. Staphylococcus epidermidis
b. Pseudomonas aeruginosa
c. Escherichia coli
d. Bacillus subtilis
i. MIXED slide of S. epidermidis and E. coli

787.

Gram Stain Procedure

794.
795.
1. Prepare slides for Gram stain as instructed above.
796.
2. Attach a clothespin to the end of the slide and hold it over the sink.
797.
3. Cover the slide with Crystal Violet stain and allow it to react for 1 minute
798.
4. Rinse the slide with a small amount of running water and remove the
excess water by gently
799.
shaking the slide.
800.
5. Cover the slide with Grams Iodine--allow it to react for 1 minute.
801.
6. Rinse the slide with water as in Step 4.
802.
7. Hold the slide at an angle and apply 1 drop of decolorizer
803.
8. Immediately rinse the slide thoroughly and remove any excess water as in
Step 4.
804.
8. Immediately rinse the slide thoroughly and remove any excess water as in
Step 4.
805.
9. Cover the slide with Safranin for 1 minute.
806.
10. Rinse the slide thoroughly.
807.
11. Blot the slide dry by placing it in between the sheets of the bibulous paper
and lightly pat
808.
with your hand.
809.
Apply immersion oil when ready to view & record in Results

section
810.
811.
812.

Procedure II

813.

Acid-fast Stain Procedure

814.
Organisms:
815.
e. Mycobacterium smegmatis (pathogenic)
816. f. Staphylococcus epidermidis

817.
818.
819.

820.
1. Prepare slides as instructed above.
821.
2. Cover the slides with a piece of paper towel the same size As the smear.
822.
3. Place clothespins at both ends of the slide to form a rack And place it on
top of the steaming water
beaker.
823.
4. Flood the slide with Carbolfuchsin stain.
824.
5. Gently steam the slide for 10 minutes, reapplying the stain
825.
as needed to prevent the slide from drying out.
826.
6. Remove the paper towel carefully with forceps and place in trash.

827.
7. Rinse the slide with a small amount of running
water until the excess stain is removed.
828.
8. Hold the slide at an angle and apply acid alcohol
by making drops at the highest part of the slide
829.
(Near the clothespin handle) and allow it to drip down the slide. Do this for
25-30 seconds.
830.
9. Rinse the slide.
831.
10. Apply several drops of Methylene Blue stain and leave for 45 seconds.
832.
11. Rinse the slide thoroughly and blot dry.
833.
Apply immersion oil when ready to view & record in Results section

834.
835.
836.
837.
838.
839.

Procedure III

841.
842.
843.

Organisms:
g. Bacillus subtilis
h. Eshericia coli

844.

840. Endospore

Stain Procedure

845.
1. Prepare slides as instructed above.
846.
2. Apply clothespins to each end of the
slide
847.
and place over a steaming beaker of
water
848.
(like acid-fast).
849.
3. Apply a piece of paper towel cut to the
size of the
850.
slide on the surface of the slide.
851.
4. Flood the paper towel with Malachite
Green stain.
852.
5. Allow the slide to steam for 5 minute
and reapply the
853.
stain as needed to prevent the paper Towel from
854.
dying out.
855.
6. Remove the paper towel gently using forceps and remove
856.
one clothespin.
857.
7. Rinse the slide with a small amount of running water.
858.
8. Hold the slide over the sink and apply Safranin and allow it
859.
to react for 1 minute.
860.
9. Rinse and blot dry.

861.
862.
863.
864.
865.

866.

Apply immersion oil when ready to view & record in Results

section

867.

868.
869.

870.
E4 Write-up
871.
Introduction paragraph: include a description of the following items from the
background
872.
Information:
873.
Gram stain, endospore stain,acid-fast and motility media
874.
Submit results from lab manual
875.
Conclusion:
876.
Use your results to describe the cellular characteristics of each organism
tested
877.
This write-up must be typed and be in your own words.

878.
879.
880.
881.
882.
883.

E4 Results

884.
Procedure I-Gram Stain
assigned microscope
#:_____________________
885.
Slide Label
886. Organism
887.
Sketch &
Indicate Color
888.
a.
889.
Staphylococcu
890.
s epidermidis
891.
b.
892.
Pseudomonas
893.
aeruginosa
894.
c.
895.
Escherichia
896.
coli
897.
d.
898.
Bacillus
899.
subtilis
900.
i.
901.
MIXED S.
902.
epidermidis and E.
coli
903.
Procedure II-Acid-fast
assigned microscope
#:_____________________
904.
Slide Label
906.
Organism
907.
Sketch

905.
908.

911.

915.

909.

e.

912.
Mycobacteriu
m smegmatis
913.
(pathogenic)
(pathogenic)

910.

914.
919.

916.
917.

921.

f.

920.
Staphylococcu
s epidermidis
918.
922.
Procedure III-Endospore Stain
#:_____________________
923.
Slide Label
924.
Organism

926.
927.

925.

929.
g.

Sketch & Color

931.

930.
Bacillus
subtilis

928.
932.
933.

assigned microscope

935.

937.

h.

936.
Escherichia
coli
934.
938.
Procedure IV-Motility Results: Draw the regions where growth was
observed.

939.
940.

Organi
sm

941.

942.

944.

947.

943.
1.)
Escherichia
coli

945.
2.)
Pseudomonas
946.
aeruginosa

948.
2.)
Pseudomonas
949.
Aerugi
nosa

950.
951.

952.

Sketch

953.
954.

955.
956.

957.
958.

959.
960.

See previous page for E4 write-up assignment.

961.
962.
963.
964.
965.
966.
967.
968.
969.
970.
971.
972.

974.

973. EXERCISE 5
CULTURING: MEDIA SELECTION AND
INOCULATION

975.

TECHNIQUES

976.
977.
Introduction:
978.
In the previous four exercises we became familiar with visual techniques used
in microbiology.
979.
You learned to describe various colony characteristics that, although not often,
may aid in the
980.
determination of a particular microbe. We also examined various types of
bacteria under the
981.
microscope to record cell morphology and any biochemical/structural
differences that could be
982.
obtained through the use of staining procedures. Although helpful,
determination of cell type and
983.
structure is limited when it comes to correctly identifying a microorganism.
There are many
984.
species that, for example, are Gram negative rods with motility. When
studying a bacterium or
985.
when trying to diagnose a disease for proper treatment, the exact species (or
even strain of that
986.
species) must first be determined.

987.
988.
For further characterization we look to various metabolic differences that are
specific to that type
989.
of bacteria. All living organisms must acquire energy from their environment.
There are
990.
differences in energy sources and metabolic pathways between groups of
bacteria. There are
991.
also ranges of environmental conditions that are particular to groups of
bacteria (pH, temperature,
992.
atmospheric requirements, etc.) where they best function (refer to E2 for
temperature differences).
993.
Knowing and manipulating these conditions provide excellent tools for
correctly identifying
994.
bacteria. The next two exercises will focus on various procedures that have
been developed to
995.
manipulate differences in energy needs and environmental condition ranges.

996.
997.
Exercise 5 will be focusing on the use of media. For solid media, agar base
(solidifying agent) is
998.
added to a broth containing nutrients that provide energy for microbes so that
they may
999.
metabolize and replicate. The ingredients that are present in the broth will
determine what groups
1000.
of microbes are able to metabolize and grow. There are some metabolic
differences among
1001.
similar microbes that can be used for identification (see differential media
below). Metabolic
1002.
differences are often observed using colorimetric tests which typically
incorporate either a pH
1003.
indicator that will change the color of the media if there is a change in pH or a
chemical that
1004.
attaches to the metabolite of interest and concentrates in the microbes
changing the normal
1005.
colony color.

1006.

1007.
In general there are three different groups of media type used in this lab:
1008.
Complex media is comprised of partially digested chemical compounds
from organic
1009.
substances such as yeast, meats, dairy products, tissues, or vegetable
materials. The
1010.
amount of each cannot be known due to differences between the organic
compounds
1011.
and the amount of digestion that has occurred. This knowledge is not
necessary when
1012.
culturing most types of microbes. This media type is useful when trying to
grow out
1013.
bacteria, or when culturing a mixed diverse sample (see E2). An example of
a complex
1014.
media is Trypticase Soy Agar (TSA).

1015.
1016.
Selective media is used to select, or isolate, specific groups of bacteria.
This is usually
1017.
based on a known environmental condition range they can tolerate that
most groups cannot.

1018.
1019.
Differential provides chemical compounds that bacteria metabolize
differently. This
1020.
difference is observed by colony or media color change once the microbe has
been
1021.
introduced to the media and allowed to grow and metabolize. Often this type
of media is
1022.
used to differentiate between similar groups of organisms with the same
morphology and biochemical resemblances.

1023.
1024.
1025.

1026.
There are several medias that are both selective and differential these include
Eosin
1027.
Methylene Blue and Mannitol Salt Agar.
1028.
Eosin Methylene Blue (EMB) is used to differentiate between gramnegative,
1029.
enteric, rods because this media primarily supports (or selects for) the
growth of
1030.
these organisms. Growth on EMB can be used to differentiate
organisms based
1031.
on their ability to ferment lactose. During the fermentation process, acid is
1032.
produced as a waste product. When grown on EMB this acid production results
1033.
in a colony color change ranging from pink to purple. In the case of E. coli and
1034.
Klebsiella pneumoniae, colonies can appear dark purple with a metallic
green
1035.
sheen. One exception to EMB selectively growing gram-negative rods is
the
1036.
gram-positive cocci Enterococcus faecalis. E. faecalis is able to grow on
this
1037.
media because it is an enteric bacterium.

1038.

1039.
Mannitol Salt Agar (MSA) contains salt that most organisms cannot tolerate
due to their
1040.
osmolarity ranges. Microbes that normally exist in an
1041.
environment with this condition, such as microbes of the skin, are
1042.
able to grow. For those halotolerant organisms, MSA is also
1043.
used to differentiate species based on their ability to ferment
1044.
mannitol. Organisms that can ferment mannitol produce an acid
1045.
by-product causing the pH indicator in the media to turn from pink
1046.
to yellow.

1047.
1048.
1049.
In addition to using color changes to differentiate bacteria,
1050.
location of growth in a special media can be useful as we have already seen
with motility media
1051.
(E4). Media can be used to determine whether a microbe is an obligate
1052.
aerobe (A), obligate anaerobe (B), or a facultative anaerobe (C) species
1053.
by observing location of growth in special media (see figure). An oxygen
1054.
gradient is formed in the oxygen requirement media once the test tube is
1055.
boiled. An aerobic environment remains in the top 1-2 cm of the test tube,
1056.
while anaerobic conditions are present below this point.

1057.
1058.
1059.
1060. When working with media it is important that you place the
1061. bacteria on the media in an informative way. When trying
1062. to simply grow bacteria on a media the best method to
1063. use is a streak-to-grow technique. With this method
1064. you are simply trying to get the bacteria on the media and you
can either use a
1065. back-and-forth motion on the plate with your loop or one
distinct streak. If you want
1066. to obtain isolated colonies for use in working with a pure
culture, than a T-streak
method would be more appropriate. The idea is to dilute or disperse the cells

1067.
so
1068.
when incubated, they will form isolated colonies that are separated to such a
degree
1069.
the colonies do not touch. This will allow viewing of the individual colonies for
any
1070.
distinguishing culture characteristics as well as for creating and working with
a pure culture.

1071.

1072.

Activities

1073.
1074.
1075.
1076.
1077.

Materials
Per team:
4 TSA plates
2 EMB plate
2 MSA plates
4 Oxygen Requirement tubes

1078.

1079.
1080.

Per table:

1081.
Test tube rack containing Escherichia coli, Staphylococcus epidermidis,
Staphylococcus aureus,
1082.
Pseudomonas aeruginosa, Enterobacter aerogenes, Enterococcus faecalis,
Klebsiella
1083.
pneumoniae, Clostridium perfringens, and Micrococcus luteus

1084.

1085.
1086.

Activity I: Inoculation and Isolation of Colonies

Isolation of a Colony: T-Streak Technique

1087.
NAME, class
1088.
1089.
1090.
1091.
1092.
1093.
1094.
below).
1095.
overlap
1096.
1097.
step 4
1098.
1099.
1100.
1101.

1102.
1103.
1104.
1105.
1106.

1107.

1. Obtain a TSA plate and label on the back your groups initials, YOUR
time, 1-4, and divide your plate by drawing a T (see below).
1. Staphylococcus epidermidis
2. Pseudomonas aeruginosa
3. Escherichia coli
4. Enterobacter aerogenes
2. Sterilize your transfer loop and allow it to cool.
3. Aseptically obtain a loop of broth from one of the cultures (see box
4. Streak the top section with closely spaced streaks that DO NOT
5. FLAME loop
6. Drag bacteria from top section into right hand section, streak as in
7. FLAME loop
8. Drag bacteria from right section to left section, streak as in step 4
9. FLAME loop
10. Invert and Incubate at 37oC

1108.
1109.
1110.
1111.
1112.
1113.
1114.

1115.

Activity II: Media Selection (MSA & EMB)

1116.
1117.
1. For each type of media plate (2 MSA & 2 EMB) label with your initials, class
day/time,
1118.
and label one set of MSA and EMB plates as a-d (see figure) and the
second set of plates e-h.
1119.
2. For each plate aseptically streak to grow with the appropriate culture.
Remember to
1120.
always practice good aseptic technique, especially between the transfers of
the different
1121.
species.

1122.
1123.
1124.
1125.
1126.
1127.
1128.
1129.
1130.
1131.

a. Staphylococcus epidermidis
b. Staphylococcus aureus
c. Micrococcus luteus
d. Enterococcus faecalis
e. Pseudomonas aeruginosa
f. Escherichia coli
g. Enterobacter aerogenes.
h. Klebsiella pneumoniae

1132.
1133.
1134.
1135.
1136.
1137.
1138.

1139.
1140.
1141.
1142.
1143.
1144.
1145.
1146.
1147.
1148.
1149.
1150.
1151.

3. Invert and incubate all 4 plates 37oC.

1152.
1153.
1154.
1155.
1156.
1157.
1158.
1159.
1160.
1161.
1162.
1163.
1164.
1165.
1166.
1167.
1168.

1169.

Activity III using media to determine O2 requirements:

1170.
1171.
1. Obtain 4 Oxygen Requirement test tubes. Label with initials, day/time, and
5-8.

1172.
1173.
2. Allow the oxygen requirement tube to cool to body temperature (it will feel
comfortably
1174.
warm to the touch). DO NOT allow your media to solidify before inoculation.
Be sure to
1175.
sterilize the full length of the loop and wire to prevent cross
contamination of the tubes.

1176.
1177.
3. Inoculate the test tube with the appropriate microbe. To do this, put the
loop with
1178.
inoculate straight down to the bottom of the tube and pull out. Repeat for all 4
species.

1179.
1180.
1181.
1182.
1183.

1.
2.
3.
4.

1184.
1185.

E5 Results:

1187.
1188.
1189.
1190.

Obtain your best T-Streak plate and observe the growth. Sketch your
results in the diagram below, and then count the total number of isolated
colonies you observe.
Total Isolated colony count: _____

1186.

1191.
1192.
1193.
1194.

Klebsiella pneumoniae
Pseudomonas aeruginosa
Escherichia coli
Clostridium perfringens

Activity I: Inoculation and Isolation of Colonies

1195.
1196.
1197.
1198.
1199.
1200.
1201.
1202.
1203.
1204.