SUBMITTED BY:

AMNA JAVED
2021-EN-06
SUBMITTED TO:

MAM QURAT-UL-AIN

EXPERIMENT NO. 7-12

ENVIRONMENTAL MICROBIOLOGY
FINAL LAB MANUAL
 EXPERIMENT NO. 07

MICROSCOPIC IDENTIFICATION OF ALGAE IN FRESH AND

POLLUTED WATER AND TO IDENTIFY THE TYPE OF ALGAE

Objective:

 To examine Algae under a microscope.

 To identify and study the microorganisms present in Polluted water algae.

RELATED THEORY

ALGAE:

Alga is a term that describes a large and incredibly diverse group of eukaryotic, photosynthetic
lifeforms. These organisms do not share a common ancestor and, hence, are unrelated
(polyphyletic).

EXAMPLES:

 Volvox

 Spirogyra

 Ulothrix

 Fucus

CHARACTERISTICS OF ALGAE:

 Algae are photosynthetic organisms

 Algae can be either unicellular or multicellular organisms

 Algae lack a well-defined body, so, structures like roots, stems or leaves are absent

 Algaes are found where there is adequate moisture.

  Reproduction in algae occurs in both asexual and sexual forms. Asexual reproduction
occurs by spore formation.

 Algae are free-living, although some can form a symbiotic relationship with other
organisms.

ALGAL BLOOM:

Algal bloom is the rapid increase in the algal population in a water body such as rivers or lakes.
Often, it is characterized by discolouration of the water and a peculiar odour

EUTROPHICATION:

Eutrophication is the process by which an entire body of water, or parts of it, becomes
progressively enriched with minerals and nutrients, particularly nitrogen and phosphorus. It has
also been defined as a "nutrient-induced increase in phytoplankton productivity.

TYPES OF ALGAE:

1. Unicellular Algae
2. Multicellular Algae
3. Filamentous Algae
4. Colonial Algae

Algae can be categorized into seven major types with distinct sizes, functions, and colors. The
different divisions include:

 Euglenophyta (Euglenoids)
 Chrysophyta (Golden-brown algae and Diatoms)
 Pyrrophyta (Fire algae)
 Chlorophyta (Green algae)
 Rhodophyta (Red algae)
 Paeophyta (Brown algae)
 Xanthophyta (Yellow-green algae)
APPARATUS:

 Slides
 Polluted water sample
 Microscope
 Pippette

PROCEDURE:

1. We took a neat and dry glass slide.

2. Poured a drop of polluted water on it from the beaker containing sample
using pipette.
3. There was no need of methylene blue dye here so we simply put the
cover strip on the drop of water.
4. And then put it in the microscope for observation.
5. We started observing from LPO. Identified the micro-organism and type
of algae nd then recorded the observation.
OBSERVATION:

RESULT:

The observed structure is related to MICROSPORA- type of algae.

 EXPERIMENT NO. 08:

Study of staining technique for microorganisms, Gram Staining

Objective;

 Examination of bacteria under microscope.

 Identification and examination of two types of bacteria i.e, gram positive and gram
negative bacteria.

Apparatus & Materials

 Microscope
 Contaminated water sample
 Hot Plate
 Pipette
 Glass slide

Chemicals

 Crystal Violet
 Iodine
 Decolorizer (95% Ethanol+5% Deionized Water)
 Safranin

Related Theory

.History

The Gram staining is one of the most crucial staining techniques in microbiology. It gets its
name from the Danish bacteriologist Hans Christian Gram who first introduced it in 1882,
mainly to identify organisms causing pneumonia.
.Initial Staining

Often the first test performed, gram staining involves the use of crystal violet or methylene blue
as the primary color. The term for organisms that retain the primary color and appear purple-
brown under a microscope is Gram-positive organisms. The organisms that do not take up
primary stain appear red under a microscope and are Gram-negative organisms. The first step in
gram staining is the use of crystal violet dye for the slide's initial staining.

.Using Iodine and Decolorization

The next step, also known as fixing the dye, involves using iodine to form crystal violet- iodine
complex to prevent easy removal of dye. Subsequently, a decolorizer, often solvent of ethanol
and acetone, is used to remove the dye. The basic principle of gram staining involves the ability
of the bacterial cell wall to retain the crystal violet dye during solvent treatment. Gram-positive
microorganisms have higher peptidoglycan content, whereas gram-negative organisms have
higher lipid content.

Initially, all bacteria take up crystal violet dye; however, with the use of solvent, the lipid layer
from gram-negative organisms is dissolved. With the dissolution of the lipid layer, gram
negatives lose the primary stain. In contrast, solvent dehydrates the gram-positive cell walls with
the closure of pores preventing diffusion of violet-iodine complex, and thus, bacteria remain
stained. The length of decolorization is a critical step in gram staining as prolonged exposure to a
decolorizing agent can remove all the stains from both types of bacteria.

.Use of counterstain

The final step in gram staining is to use basic fuchsin stain to give decolorized gram-negative
bacteria pink color for easier identification. It is also known as counterstain. Some laboratories
use safranin as a counterstain; however, basic fuchsin stains gram-negative organisms more
intensely than safranin. Similarly, Hemophilus spp., Legionella app, and some anaerobic bacteria
stain poorly with safranin

.
Procedure

1. We made a slide of cell sample to be stained. We heated the slide to fix

the sample to the slide by carefully passing the slide with a drop or
small piece of sample on it through a bunsen burner three times.

2. Then, added the primary stain (crystal violet) to the sample/slide and
incubate for 1 minute. Rinsed slide with a gentle stream of water for a
maximum of 5 seconds to remove unbound crystal violet.

3. After this, we added Gram's iodine for 1 minute- this is a mordant, or an

agent that fixes the crystal violet to the bacterial cell wall.

4. Again, rinsed the sample/slide with acetone or alcohol for ~3 seconds

and rinse with a gentle stream of water. The alcohol will decolorize the
sample if it is Gram negative, removing the crystal violet. However, if
the alcohol remains on the sample for too long, it may also decolorize
Gram positive cells.

5. By adding the secondary stain, safranin, to the slide and incubate for 1
minute. Wash with a gentle stream of water for a maximum of 5
seconds. If the bacteria is Gram positive, it will retain the primary stain
 (crystal violet) and not take the secondary stain (safranin), causing it to
look violet/purple under a microscope. If the bacteria is Gram negative,
it will lose the primary stain and take the secondary stain, causing it to
appear red when viewed under a microscope.

OBSERVATIONS:

RESULT:

We have observed the gram positive and gram negative bacteria under the
microscope. The pink colour represent gram negative and purple colour
represent the gram positive bacteria.
 EXPERIMENT NO. 10:

Determination of Total Coliform & Fecal Coliform by MPN Method.

Objective:

The main objective of this experiment is to estimate Total coliform & Fecal coliform by MPN
method.

Apparatus Required:

 Autoclave
 Test tubes
 Incubator
 Sample bottles
 Fermentation tubes with inverted vials
 Dilution bottles
 Pipettes and pipette stand
 Fermentation tubes with caps
 Inverted vials
 Sterile metal loop 3 mm in diameter
 Sprit Lamp

Chemicals Required:

 Lactose broth
 Distilled water
 EC (Escherichia coli) broth

Related Theory:
Total Coliform:The Coliform group comprises all aerobic and facultative anaerobic, gram
negative, non-spore forming, rod shaped bacteria that ferment lactose with gas and acid
formation within 48 hours at 350C.
 Fecal Coliform:

Coliform bacteria are a natural part of the microbiology of the intestinal tract of warm
blooded mammals, including man. The total coliform group is relatively easy to culture in
the lab, and therefore, has been selected as the primary indicator bacteria for the presence
of disease causing organism. Coliform bacteria are generally not harmful to human health
however the total coliform group includes fecal coliforms and E. coli. As such, the
presence of coliform organisms can indicate the possibility of pollution by human or
animal waste. Health based guideline for Total coliform is 0 organisms/100mL & must
not be detectable in any 100ml sample. (Each person discharges from 100 to 400 billion
fecal coliform organisms per day).

Lactose Broth:

Lactose Broth is used for the detection of coliform bacteria in water, foods,
and dairy products as per Standard Methods.
 EC (Escherichia coli) broth:

EC Medium is used for detection of coliforms during bacteriological examination of

water, milk and medium for optimal recovery of fecal coliforms is required.

Procedure:

A relatively easy water quality test uses the Most Probable Number, or MPN, method, and is
composed of three phases (Presumptive phase, Confirmatory phase & completed phase):

Procedure for presumptive phase

Dilution
We will prepare three dilutions of given sample upon quality of pathogens present in water.
Ideally we prepare 2 dilutions. The way in which dilutions are made is:
 First dilution was our sample and we named it as 10.
 Second dilution was one in which we took 10ml of sample and added 90ml of water in it,
and called it 1.
 Third dilution was one in which we took 10ml of second dilution and further added 90ml
of water in that, and was called 0.1.

Sterilization
There are 3 sets of fermentation tubes and each set comprises of 15 tubes with small vials, but
small vials are in inverted position.
Then sterilize all these tubes using autoclave at 121°C for 15 minutes. This is standard method to
remove contaminants.
 15 test tubes contain one type of broth lactose broth.
 Place filled and inverted vials in autoclave.
 Place them in incubator for 48 hours at 35 C.

Procedure for confirmed and complete phase

After incubation we use only positive tubes for further testing and negative tubes are discarded.
Tubes having lifted vials are considered to be positive tubes.

 I took the positive tubes with bubbles trapped in vials and do further testing.
  I took a wire loop, sterilize it on flame, dip it in the positive tube and inoculate it in
BGLB and do the same with EC broth.
 Then, inoculated all tubes like this and place these samples in incubator for 24 hours.
BGLB at 35 °C and fecal at 44°C. (Confirmed phase should be 35°C because total
coliforms grow only at this temperature and fecal coliforms grow specifically at 44°C.)
 Then saw positive tubes from there and did the calculations by using formula given as.

Environmental Significance:

Drinking water testing:

 Testing for all individual pathogens is impractical and expensive. Instead, the EPA has
designated total coliform bacteria as a standard to determine bacterial safety of water.
 Fecal Coliform bacteria indicate the presence of sewage contamination of a waterway and
the possible presence of other pathogenic organisms.

Calculation:
 Lactose broth (positive tubes):

Serial no. mL of sample No. of +ve tubes No. of –ve tubes

1 10 5 0
2 1 3 2
3 0.1 2 3
no . of positive tubes ×100
MPN/100ml ¿
√ ml of sample ∈negative tubes ×ml of sample ∈all tubes
10 × 100
¿ = 88.51mpn\100 ml
√2.3 × 55.5
 EC broth (positive tubes):

Serial no. ml of sample No. of +ve tubes No. of –ve tubes

1 10 2 3
2 1 1 2
3 0.1 1 1

no . of positive tubes ×100

MPN/100ml ¿
√ ml of sample ∈negative tubes ×ml of sample ∈all tubes
4 ×100
¿
√32.1 ×55.5
Fecal Coliform = 9.476mpn\100ml

BGLB: (TOTAL COLIFORM)

Serial no. ml. of sample No. of +ve tubes No. of –ve tubes
1 10 3 2
2 1 2 1
3 0.1 1 1

no . of positive tubes ×100

MPN/100ml ¿
√ ml of sample ∈negative tubes ×ml of sample ∈all tubes
6 × 100
¿ = 17.53mpn\100
√21.1 ×55.5

RESULTS:

We found out the MPN\100mls of:

Lactose broth=88.51
 EC broth=9.476
Total coliform=17.53

EXPERIMENT NO. 09:

Preparation of Pure Culture Medium

OBJECTIVES:

• Preparation of microbial cultures that will be used to determine the type of organism, its
abundance in the sample being tested, or both

APPARATUS:

 Petri dishes
 Incubator
 Culture media
 Burner
 Inoculating loop
 Bunsen Burner
 Broth

RELATED THEORY:

INTRODUCTION:

• A microbial culture, is a method of multiplying microorganisms by letting them

reproduce in predetermined culture media under controlled laboratory conditions.

• An artificial culture media must provide similar environmental and nutritional conditions
that exist in the natural habitat of a bacterium.

• A culture medium contains water, a source of carbon & energy, source of nitrogen, trace
elements and some growth factors. The pH of the medium must be set accordingly.
Purpose of culturing:

• Isolation of bacteria.

• Properties of bacteria i.e. culturing bacteria is the initial step in studying its morphology and its
identification.

• Maintenance of stock cultures.

• Estimate viable counts.

• To test for antibiotic sensitivity.

• To create antigens for laboratory use.

Agar Plate:

• An agar plate is a petri dish that contains a growth medium (typically agar + nutrients)
used to culture microorganisms.

• Agar plates may also indicator plates, in which the organisms are not selected on the
basis of growth but are instead distinguished by a color change in some colonies,
typically caused by the action of an enzyme on some compounds added to the medium.

• Selective growth compound may also be added to the media such as antibiotics.

Procedure:

 I took 45 grams of Tripric broth and dissolved it in 1 liter of distilled water.

 I sterilized the culture media in an autoclave for 15 minutes under 15-pound pressure at
121 degrees.
 After the media was sterilized I took a petri dish and poured prepared culture media.
 Then I put the petri dish under the sterilized conditions.
 I waited for 10 to 15 minutes until the culture media was solidified.
 Then I applied the streaking technique on solid surface of culture media and incubated
the petri dish at 37 degrees for 24 hours.
I repeated this same procedure for 49.53 grams of macconkey agar
OBSERVATIONS:

EXPERIMENT NO. 10:

To determine the quality of milk by Methylene Blue Reduction Test
OBJECTIVE:
• This test is performed to check the bacteria contamination in milk. It will visually
indicate the presence of bacteria in a given milk sample, and it will indicate the level of
milk quality.

APPARATUS:
 Milk sample
 Pipette
 Burner
 Test tubes
 Test tubes rack
 Water bath

RELATED THEORY
INTRODUCTION:
• Methylene Blue Reduction Test also known as MBR test. It is a qualitative test for
milk, it used to check the quality of raw and pasteurized milk.
• The Methylene Blue Reduction Test is based on the fact that in the presence of oxygen
the methylene blue solution forms blue color, and it will lose the color as the oxygen is
depleted.
• The bacteria present in the milk will ferment lactose (milk sugar) to form lactic acid,
during this fermentation process the oxygen is used up, which causes in depletion of
oxygen in milk, and electrons are released. These electrons react with the methylene blue
solution. As a result, it decolorizes the methylene blue.
• Mainly bacteria are responsible for the oxygen consumption in milk. It is estimated that
assumed that the greater the number of bacteria in milk, the quicker will the oxygen be
consumed. The total number of microorganisms in milk.

PRINCIPLE:
• Milk has sufficiently low redox potential which reduces the methylene blue
immediately. During the milking, cooling, dumping the oxidation-reduction
potential of milk is increased to +0.3V. At this point, the methylene blue
remains in an oxidized state.
• When the bacterial cells are started to increase their numbers in milk it
consumes more dissolved oxygen from the milk, as a result, the oxygen gets
depleted. Then the Methylene Blue starts acting as an electron acceptor
instead of oxygen. The methylene blue gets reduced due to the decreases of
oxidation-reduction potential from + 0.06 to 0.01 V.
• The double-bonded nitrogen atom of Methylene Blue dye accepts 1 atom of
hydrogen as a result the dye is converted into a colorless state. The greater is
the number of microorganisms in milk, the greater is the metabolic activity
and the faster is the reduction of methylene blue.
PROCEDURE:

 I took four test tubes and added 10 ml of milk sample in each of the test tube.
 Then I added 1ml of methylene blue indicator using a pipet and mixed it thoroughly.
 Then I closed the test tubes with caps and placed it in the water bath at 37 degrees.
 I observed the decolorization of milk after every 30 minutes time interval and recorded
the results.

OBSERVATIONS:
TABLE:

SERIAL SAMPLE INITIAL TOTAL CHANGES

NO. TIME TIME
1 A 10:04-10:34 30 minutes Decolourization
10:04-11:08 1 hour start
10:04-11:42 1:30 hour
10:04-12:12 2 hours
2 B 10:04-10:34 30 minutes Decolourization
10:04-11:08 1 hour start
10:04-11:42 1:30 hour
10:04-12:12 2 hours
3 C 10:04-10:34 30 minutes Decolourization
10:04-11:08 1 hour start
10:04-11:42 1:30 hour
10:04-12:12 2 hours
4 D 10:04-10:34 30 minutes Decolourization
10:04-11:08 1 hour start
10:04-11:42 1:30 hour
10:04-12:12 2 hours

OPEN ENDED LAB:

 A WASTEWATER TREATMENT PLANT IS FACING PROBLEM DUE
TO SLUDGE BULKING

TO VALIDATE THE CLAIM:

The plant engineer is asserting that different microorganisms are the cause of this
problem.

OBJECTIVE:

To find the filamentous structure of algae in sludge bulking.

APPARATUS:

 Slides
 Sample of sludge
 Microscope

RELATED THEORY:

SLUDGE BULKING:

Sludge bulking is the most common solids settling problem in wastewater treatment plants,
which is caused by the excessive growth of filamentous bacteria extending outside the flocs,
resulting in decreasing the wastewater treatment efficiency and deteriorating the water quality in
the effluent.

In treatment of sewage one process used is the activated sludge process in which air is passed
through a mixture of sewage and old sludge to allow the micro-organisms to break down the
organic components of the sewage. Sludge is continually drawn off as new sewage enters the
tank and this sludge must then be settled so that the supernatant (the remaining liquid) can be
separated to pass on to further stages of treatment.
GROWTH OF FILAMENTOUS BACTERIA:

Sludge bulking occurs when the sludge fails to separate out in the sedimentation tanks. The main
cause of sludge bulking is the growth of filamentous bacteria.

Filamentous microorganisms grow in long strands that have much greater volume and surface
area than conventional floc and are very slow to settle. Under certain growing conditions,
filamentous organisms predominate. There is little robust scientific evidence that can be used to
avoid sludge bulking but what there is indicates that over-loading works, having a carbohydrate-
rich input, and having too low a recycle rate may all contribute.

CAUSE OF BULKING:

The cause of bulking is normally due to either excessive filamentous growth or micro-organisms
producing extracellular such as polysaccharide.

There are a number of short-term control measures which include biocide addition, use of
flocculating chemicals or increasing the RAS flow. These measures generally treat the symptoms
and not the underlying cause of the problem. The production of extracellular material and
excessive filamentous micro-organisms have been related to nutrient deficiency, low dissolved
oxygen and configuration of the aeration basin. This article deals with the control of activated
sludge bulking based on these causes.

PROCEDURE:

 A sample of sludge was provided to observe the filamentous structure.

  I placed the sample on the slide.
 Then, I covered the slide with a cover slip because the sample was wet.
 I placed the prepared slide on the microscope.
 And in the last, I observed the following structures.

OBSERVATIONS:

Result:

Here is the filamentous structure of sludge bulking under the microscope.

PRECAUTIONS:

 Wear mask and gloves while preparing sample slide.

 Do not make contact with your face or with your fellow members.
 Wash hands properly after experiment is completed.
 Use different instruments carefully.
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