G.

Bacteriology
Lab Manual
 Exp-1: Preparation of Bacteriological Media
Principle:
Microbiology research depends largely on the ability to grow and maintain microorganisms in the
laboratory, and this is possible only if suitable culture media are available. A culture medium is a solid
or liquid preparation used to grow, transport, and store microorganisms. To be effective, the medium
must contain all the nutrients the microorganism requires for growth. Specialized media are essential in
the isolation and identification of microorganisms, the testing of antibiotic sensitivities, water and food
analysis, industrial microbiology, and other activities. Although all microorganisms need sources of
energy, carbon, nitrogen, phosphorus, sulfur, and various minerals, the precise composition of a
satisfactory medium depends on the species one is trying to cultivate because nutritional requirements
vary so greatly. Knowledge of a microorganism’s normal habitat often is useful in selecting an
appropriate culture medium because its nutrient requirements reflect its natural surroundings. Frequently
a medium is used to select and grow specific microorganisms or to help identify a particular species.
The mixture of necessary nutrients can be used as a liquid medium, or a solidifying agent can be added.
N-agar consists of 5g/L peptone, 3g/L beef extract, 5g/L sodium chloride and 15g/L agar.
Dissolve all the ingredients in 1L. Autoclave the media at 121 C, 12 lb pressure for 15 minutes. Both
N-agar and N-Broth are rich in nitrogen, carbon and salts to meet the requirement of bacterial growth.
For convenience both N-agar media are supplied as premixed preparations.
TYPES OF MEDIA
Basic Classification of Media Types
Defined (synthetic): A medium in which all chemical components are
known is a defined or synthetic medium. It can be in a liquid form
(broth) or solidified by an agent such as agar. E.g: M9 minimal
medium.
Chemical Composition Complex: Media that contain some ingredients of unknown chemical
composition are complex media. Complex media are nutrient-rich
substances that contain a variety of undefined components such as
peptones, extracts, and digests from animal or plant sources, blood.
Such media are very useful, as a single complex medium may be
sufficiently rich to meet all the nutritional requirements of many
different microorganisms. In addition, complex media often are
needed because the nutritional requirements of a particular
microorganism are unknown, and thus a defined medium cannot be
constructed. E.g: N-broth, MacConkey Agar.
Liquid: A liquid medium lacks a solidifying agent and is called a
broth medium.
Solid: A broth medium supplemented with a solidifying agent called
agar results in a solid or semisolid medium. Agar, an extract of
seaweed, is a complex carbohydrate composed mainly of galactose,
Physical Nature
and is without nutritional value. Agar serves as an excellent
solidifying agent because it liquefies at 100°C and solidifies at 40°C.
Because of these properties, organisms, especially pathogens, can be
cultivated at temperatures of 37.5°C or slightly higher without fear of
the medium liquefying. A completely solid medium requires an agar
concentration of about 1.5 to 1.8%.
Semi solid: A medium containing concentration of less than 1% agar
results in a semisolid medium.
Supportive (general purpose) Media: Tryptic soy broth and tryptic
 soy agar are called general purpose or supportive media because they
sustain the growth of many microorganisms.
Enriched Media: Blood and other special nutrients may be added to
supportive media to encourage the growth of fastidious microbes.
These specially fortified media (e.g., blood agar) are called enriched
media.
Function Selective Media: Favor the growth of particular microorganisms. Bile
salts or dyes such as basic fuchsin and crystal violet favor the growth
of gram-negative bacteria by inhibiting the growth of gram-positive
bacteria; the dyes have no effect on gram-negative organisms. E.g.,
MacConkey agar, Salmonella Shigella agar.
Differential Media: Media that distinguish among different groups of
microbes and even permit tentative identification of microorganisms
based on their biological characteristics. MacConkey agar is both
differential and selective. Since it contains lactose and neutral red dye,
bacteria that catabolize lactose by fermenting it release acidic waste
products that make colonies appear pink to red in color. These are
easily distinguished from colonies of bacteria that do not ferment
lactose.
Figure 1: Types of Media

Figure 2: Figure shows the major types of growth media and their purpose
Materials:
 Flasks
 Graduating cylinder
 Incubator
 Weighing balance
 Beaker
 Distilled water
 Autoclave
 Petri plates
 Spirit lamp
 Cotton
  70% ethanol

Composition of N-Agar/L-Broth (1 Liter):

 5g Peptone
 5g NaCl
 3g Beef Extract
 15g agar

(Agar is added only when N-Agar is to be prepared. In case of N broth, agar is not included in the
composition. The components are dissolved in distilled water followed by autoclaving at 121 C at 15 lb
pressure for 15 minutes)

Methodology:
1. The required components of growth medium were weighed by using weighing balance.
2. All the components of N-Agar or N-Broth were mixed in 100ml distilled water in a flask.
3. Flask was covered properly with a cotton plug and then with aluminum foil.
4. Media was autoclaved at 121 C at 15 lb pressure for 15-20 minutes.
Note: Do not open the autoclave until the temperature drops to 80 degree.
6. Flask was taken out by the help of heat resistant gloves.
7. Then flask was placed on a sterile working bench.
Note: Do not open the flask until required or experiment is to be performed.
8. When the media temperature became luke warm (almost 45C to 50 C) then media was poured in
sterile petri plates.
9. Plates were left on bench top to solidify. To check the sterility of media, plates were placed in
incubator for 24 hours at 37 C. After 24 hours of incubation no growth was observed it means the
plates were sterile and it can be used for experimentation.
 Exp-2: Isolation of Bacteria from Waste Water sample by Spread Plate Technique
Principle:
Microorganisms are ubiquitous. They are found in soil, air, water, food, sewage, and on body surfaces.
In short, every area of our environment is replete with them. The microbiologist separates these mixed
populations into individual species for study. A culture containing a single unadulterated species of cells
is called a pure culture (Another definition of Pure culture: A culture that contains only a single
species or strain of microorganism, without any other contaminant). To isolate and study
microorganisms in pure culture, the microbiologist requires basic laboratory apparatus and the
application of specific techniques. In nature, microbial populations do not segregate themselves by
species but exist with a mixture of many other cell types. In the laboratory, these populations can be
separated into pure cultures. These cultures contain only one type of organism and are suitable for the
study of their cultural, morphological, and biochemical properties. In this experiment, you will first use
one of the techniques designed to produce discrete colonies. Colonies are individual, macroscopically
visible masses of microbial growth on a solid medium surface, each representing the multiplication of a
single organism. Microorganisms are abundant in nature so for isolation of microorganism, dilution of
sample must be done to lower down the microbial load and to isolate discrete colonies.
There are 3 main techniques of isolation of bacteria
1. Spread Plate Method
2. Poured Plate Method
3. Streak Plate Method
The spread-plate technique requires that a previously diluted mixture of microorganisms be used. During
inoculation, the cells are spread over the surface of a solid agar medium with a sterile, L-shaped bent
glass rod. The spread plate spreads the cells on the surface of the agar. For the spread plate, a small
volume of a diluted mixture containing around 30 to 300 cells (25 to 250 cells if examining microbes in
food or water samples) is transferred to the center of an agar plate and spread evenly over the surface
with a sterile bent rod. The dispersed cells develop into isolated colonies.

Materials:
 N-agar plates
 Waste Water sample
 L-shaped spreader
 Beaker
 Bunsen Burner
 Match box
 Micropipettes
 Micropipettes tips
 Test tubes
 Test tubes stand
 Distilled water or Normal Saline
 Incubator
 Autoclave
  Measuring cylinder
 Discarding box
 70% Ethanol
 95% Ethanol
 Cotton
Methodology:
1. Sample was serially diluted. 9 test tubes were taken and 9ml distilled water was added in each
tube. All the tubes were sterilized by autoclaving. Test tubes were properly labelled.
(Note: If sample is in solid form then measure in grams, if it’s in liquid form then measure in ml)

2. Then 1ml of waste water sample was dispensed in the first tube by the help of micropipette and
mixed well.

3. From first test tube 1ml solution was transferred to the second test tube and properly mixed. This
process was done till the last test tube and from the last (9 th) test tube 1ml of solution was
discarded. Serial dilution from -1 to -9 was prepared.

4. The bent glass rod was placed into a beaker and was added a sufficient amount of 95% ethyl
alcohol to cover the lower, bent portion.

5. Appropriately labelled N. agar plates were placed on the working area (bench top). With a sterile
pipette, 50l or 100l of sample from -3, -4, -5 dilutions were placed on the center of their
respective plate.

6. Removed the glass rod from the beaker, and passed it through the Bunsen burner flame with the
bent portion of the rod pointing downward to prevent the burning alcohol from running down
your arm. Allowed the alcohol to burn off the rod completely. Cooled the rod for 10 to 15
seconds.

7. The Petri dish cover was removed. The sterile bent rod was slightly touched on the surface of the
agar and moved back and forth. The Petri dish was turned manually and sample was spread with
the sterile bent glass rod. The sample was spread over the agar surface evenly in all directions.
The cover of petri dish was re-placed. The rod was immersed in alcohol and re-flamed.

8. The petri dish was incubated at 37C for 24 hours. After 24 hours results was observed.

Results:
After the completion of incubation period different colonies were observed on the agar plates and
colony forming unit per ml was also calculated.
 Figure 1: Serial Dilution

Figure 2: Spread-Plate Technique

Figure 3: Result of spread plate method after incubation

 Exp-3: Isolation of Bacteria from soil sample by Poured Plate Technique
Principle:
Pour-plate and spread-plate techniques are similar in that they both dilute a sample of cells before
separating them spatially. They differ in that the spread plate spreads the cells on the surface of the
agar, whereas the pour plate embeds the cells within the agar. The pour plate is extensively used with
procaryotes and fungi. The original sample is diluted several times to reduce the microbial population
sufficiently to obtain separate colonies when plating. Then small volumes of several diluted samples
are mixed with liquid agar that has been cooled to about 45°C, and the mixtures are poured immediately
into sterile culture dishes. Most bacteria and fungi survive a brief exposure to the warm agar. Each cell
becomes fixed in place to form an individual colony after the agar hardens. Like the spread plate, the
pour plate can be used to determine the number of cells in a population. For both methods, the total
number of colonies equals the number of viable microorganisms in the sample that are capable of
growing in the medium used. Colonies growing on the surface also can be used to inoculate fresh
medium and prepare pure cultures.

Materials:
 N-agar media
 Soil Sample
 Bunsen Burner
 Match box
 Micropipettes
 Micropipettes tips
 Test tubes
 Test tubes stand
 Distilled water or Normal Saline
 Incubator
 Autoclave
 Measuring cylinder
 Discarding box
 70% Ethanol
 Cotton

Methodology:
1. Serial dilution of soil sample was prepared as described in the previous experiment.

2. N- Agar medium was prepared in three different flasks and autoclaved as per SOP.

3. When the temperature of media cooled down to 45C to 50 C then 1ml of sample from -3, -4, -5
dilutions were dispensed in each of N- agar flask and was gently shaken to mix the sample and
medium properly, then poured it into the sterile petri plates. Plates were left on the bench top to
solidify.

4. Then these plates were incubated at 37C for 24 hours. After 24 hours results were observed.
Results:
After the completion of incubation period different colonies were observed on the agar plates and
colony forming unit per ml was also calculated.

Figure 1: Pour-Plate Technique

 Exp-4: Purification of Bacteria by Quadrant Streaking Method
Principle:
The techniques commonly used for isolation of discrete colonies initially require that the number of
organisms in the inoculum be reduced. The resulting diminution of the population size ensures that,
following inoculation, individual cells will be sufficiently far apart on the surface of the agar medium to
separate the different species. The following are techniques that can be used to accomplish this
necessary dilution:

Materials:
 N-agar media
 Bunsen Burner
 Match box
 Incubator
 Autoclave
 Measuring cylinder
 Discarding box
 70% Ethanol
 Cotton
 Inoculating loop

Methodology:
1. The streak-plate method is a rapid qualitative isolation method. It is essentially a dilution
technique that involves spreading a loopful of culture over the surface of an agar plate.

2. A loopful of culture was placed on the agar surface in Area 1. Loop was flamed, cooled by
touching an unused part of the agar surface close to the periphery of the plate, and then it was
dragged rapidly several times across the surface of Area 1.

3. Loop was re-flamed and cooled, and Petri dish was turned 90°. Then loop was touched on a
corner of the culture in Area 1 and was dragged several times across the agar in Area 2. Note:
The loop should never enter Area 1 again.

4. Loop was re-flamed and cooled, again dish was turned at 90°. Area 3 was streaked in the same
manner as Area 2.

5. Without re-flaming the loop, the dish was again turned at 90° and then culture was dragged from
a corner of Area 3 across Area 4, using a wider streak. It was made sure that loop did not touch
any of the previously streaked areas. The flaming of the loop at the points indicated to dilute
the culture so that fewer organisms were streaked in each area that would result in the
final desired separation.

6. The petri dish was incubated at 37C for 24 hours. After 24 hours results was observed.
Results: Discrete colonies were observed after the 24 hours of incubation

Figure: Quadrant streaking Method

Figure: Quadrant streaked plate inoculation with Serratia marcescens

 Exp-5: Gram Staining
Principle:
Differential staining requires the use of at least four chemical reagents that are applied sequentially to
a heat-fixed smear. The first reagent is called the primary stain. Its function is to impart its color to all
cells. The second stain is a mordant used to intensify the color of the primary stain. In order to
establish a color contrast, the third reagent used is the decolorizing agent. Based on the chemical
composition of cellular components, the decolorizing agent may or may not remove the primary stain
from the entire cell or only from certain cell structures. The final reagent, the counterstain, has a
contrasting color to that of the primary stain. Following decolorization, if the primary stain is not
washed out, the counterstain cannot be absorbed and the cell or its components will retain the color of
the primary stain. If the primary stain is removed, the decolorized cellular components will accept the
contrasting color of the counterstain. In this way, cell types or their structures can be distinguished
from each other on the basis of the stain that is retained. The most important differential stain used in
bacteriology is the Gram stain, named after Dr. Hans Christian Gram. It divides bacterial cells into
two major groups, gram-positive and gram-negative, which makes it an essential tool for
classification and differentiation of microorganisms. This classification depends upon the composition
of cell wall of bacteria. In gram negative cells, the alcohol increases the porosity of the cell wall by
dissolving the lipids in the outer layers. Thus, the CV-I complex can be more easily removed
from the thinner and less highly cross-linked peptidoglycan layer. Therefore, the washing-out
effect of the alcohol facilitates the release of the unbound CV-I complex, leaving the cells colorless or
unstained. The much thicker peptidoglycan layer in gram-positive cells is responsible for the
more stringent retention of the CV-I complex, as the pores are made smaller due to the
dehydrating effect of the alcohol. Thus, the tightly bound primary stain complex is difficult to
remove, and the cells remain purple.

Materials:
 24 hours fresh culture
 Bunsen burner
 Inoculating loop or needle
 Staining tray
 Glass slides
 Bibulous paper
 Microscope
 Primary Stain: Crystal Violet
 Mordant : Gram’s Iodine
 Decolorizer : Ethanol or Acetone
 Counterstain/Secondary Stain: Safranin
 Immersion oil

Methodology:
Smear Preparation:
1. A neat, clean and grease free glass slide was taken.
 2. The smear was prepared by placing a drop of autoclaved distilled water on the glass slide.
3. Bacterial culture was transferred on glass slide with the help of inoculating loop. Then the smear
was prepared by rotating the loop in one direction either clockwise or anticlockwise.

4. After that, smear was air dried and heat fixed in the usual manner.

Gram Staining:
1. The smear was then gently flooded with primary stain i.e. crystal violet for 1 minute. Gently
washed with under the tap water.

2. Then the slide was flooded with mordant i.e. gram’s iodine for 1 min and washed it under tap
water carefully.

3. After that it was decolorized by the decolorizer i.e. 95% ethanol. Reagent was added drop by
drop until the alcohol runs almost clear, showing only a blue tinge. Slide was gently rinsed with
tap water.

4. Then it was stained with counter stain or secondary dye i.e. safranin for 45 seconds and carefully
washed under tap water. Slide was blot dried with bibulous paper and examined with under the
microscope at 100X oil immersion lens.

Interpretation:
 Gram positive bacteria will stain Blue/Purple
 Gram negative bacteria will stain Pink/Red

Results:
Under the microscope 100X oil immersion lens gram positive cocci were observed.
Figure: Microscopic observation of cells following steps in the Gram staining procedure

Figure: Gram positive Cocci

 Exp-6: Use of MacConkey Agar for isolation and identification of
Enterobacteriaceae
Principle:
MacConkey agar (MAC) was the first solid differential media to be formulated which was developed at
20th century by Alfred Theodore MacConkey. MacConkey agar is a selective and differential media
used for the isolation and differentiation of non-fastidious gram-negative rods, particularly members of
the family Enterobacteriaceae and the genus Pseudomonas (which is also a notorious agent of
nosocomial infections- hospital-acquired infections).
MacConkey agar is used for the isolation of gram-negative enteric bacteria and the differentiation of
lactose fermenting from lactose non-fermenting gram-negative bacteria. Pancreatic digest of gelatin
and peptones (meat and casein) provide the essential nutrients, vitamins and nitrogenous factors
required for growth of microorganisms. Lactose monohydrate is the fermentable source of
carbohydrate. The selective action of this medium is attributed to crystal violet and bile salts, which are
inhibitory to most species of gram-positive bacteria. Sodium chloride maintains the osmotic balance in
the medium. Neutral red is a pH indicator that turns red at a pH below 6.8 and is colorless at any pH
greater than 6.8. Agar is the solidifying agent.
The inhibitory action of crystal violet on the growth of gram positive organisms allows the isolation of
gram-negative bacteria. Incorporation of the carbohydrate lactose, bile salts, and the pH indicator neutral
red permits differentiation of enteric bacteria on the basis of their ability to ferment lactose. On this
basis, enteric bacteria are separated into two groups:
a. Coliform bacilli produce acid as a result of lactose fermentation. The bacteria exhibit a red coloration
on their surface. Escherichia coli produce greater quantities of acid from lactose than other coliform
species. When this occurs, the medium surrounding the growth also becomes pink because of the action
of the acid that precipitates the bile salts, followed by absorption of the neutral red.
b. Dysentery, typhoid, and paratyphoid bacilli are not lactose fermenters and therefore do not
produce acid. The colonies appear tan and frequently transparent.

Materials:

 MacConkey agar plates

 Sample (to be checked) can be waste water, soil, blood, sputum, etc.
 Beaker
  Bunsen Burner
 Match box
 Micropipettes
 Micropipettes tips
 Incubator
 Autoclave
 Measuring cylinder
 Discarding box
 70% Ethanol
 95% Ethanol
 Cotton
 Inoculating loop
Protocol:
1. 50 grams of dehydrated MacConkey media was suspended in 1000 ml distilled water.

2. Media was sterilized by autoclaving at 15 lbs pressure, (121°C) for 15 minutes.

3. Media was poured in sterile petri plates.

4. Waste water sample dilutions were prepared and then spread on MacConkey plates.

5. Medical samples were tested by using an inoculating loop, touching the sample and directly streaking on
the plates.

6. Plates were incubated at 37C for 24 hours.

7. After 24 hours of incubation results were observed.

Results Interpretation:
Lactose fermenting strains grow as red or pink and may be surrounded by a zone of acid precipitated
bile. The red color is due to production of acid from lactose, absorption of neutral red and a subsequent
color change of the dye when the pH of medium falls below 6.8.
Lactose non-fermenting strains such as Shigella and Salmonella are colorless and transparent and
typically do not alter appearance of the medium. Yersinia enterocolitica may appear as small, non-
lactose fermenting colonies after incubation at room temperature.
Results:
Pink color colonies were observed on MacConkey plates after 24 hours of incubation.
Exp-7: Determining the biochemical activities of microorganisms for identification-
Catalase test
Principle
During aerobic respiration, microorganisms produce hydrogen peroxide and, in some cases, an
extremely toxic superoxide. Accumulation of these substances will result in death of the organism unless
they can be enzymatically degraded. These substances are produced when aerobes, facultative
anaerobes, and microaerophiles use the aerobic respiratory pathway, in which oxygen is the final
electron acceptor, during degradation of carbohydrates for energy production. Organisms capable of
producing catalase rapidly degrade hydrogen peroxide as illustrated:

Aerobic organisms that lack catalase can degrade especially toxic superoxides using the enzyme
superoxide dismutase; the end product of a superoxide dismutase is H 2O2, but this is less toxic to the
bacterial cells than are the superoxides. The inability of strict anaerobes to synthesize catalase,
peroxidase, or superoxide dismutase may explain why oxygen is poisonous to these microorganisms. In
the absence of these enzymes, the toxic concentration of H2O2 cannot be degraded when these organisms
are cultivated in the presence of oxygen. If catalase is present, the chemical reaction mentioned is
indicated by bubbles of free oxygen gas (O2 ). This is a positive catalase test; the absence of bubble
formation is a negative catalase test. Figure 30.1 shows the results of the catalase test using (a) the tube
method, (b) the plate method, and (c) slide method.

Materials:
 28 to 48 hours culture plates
 3% Hydrogen peroxide
 Glass slides
 Bunsen burner
 Tooth picks
 Distilled water
 Beaker
 Micropipette
 Micropipette tips (1ml)
 Match box
 Ethanol
 Tissue paper
 Cotton
Methodology:
Slide Method:
1. Slides were labelled properly.
2. Sterile tooth pick was used to collect a small sample of test organism from the culture plate and
transfer it to the appropriately labeled slide.
3. Slide was placed in petri dish.
4. One drop of 3% hydrogen peroxide was placed on the slide. (Do not mix). Cover of petri dish was
placed on the petri dish to contain any aerosols.
5. The slide was observed for immediate presence of bubble formation.
Results:
The immediate presence of bubble formation was observed indicating a positive result.

Figure: Slide Method

Exp-8: Determining the biochemical activities of microorganisms for identification-
Oxidase test
Principle:
Oxidase enzymes play a vital role in the operation of the electron transport system during aerobic
respiration. Cytochrome oxidase catalyzes the oxidation of a reduced cytochrome by molecular oxygen
(O2), resulting in the formation of H2O or H2O2. Aerobic bacteria, as well as some facultative anaerobes
and microaerophiles, exhibit oxidase activity. The oxidase test aids in differentiation among members of
the genera Neisseria and Pseudomonas, which are oxidase positive, and Enterobacteriaceae, which are
oxidase-negative. The ability of bacteria to produce cytochrome oxidase can be determined by the
addition of the test reagent p-aminodimethylaniline oxalate to colonies grown on a plate medium. This
light pink reagent serves as an artificial substrate, donating electrons and thereby becoming oxidized to a
blackish compound in the presence of the oxidase and free oxygen. Following the addition of the test
reagent, the development of pink, then maroon, and finally dark purple coloration on the surface of the
colonies is indicative of cytochrome oxidase production and represents a positive test. No color change,
or a light pink coloration on the colonies, is indicative of the absence of oxidase activity and is a
negative test. The filter paper method may also be used and is described in this experiment.
Materials:
 28 to 48 hours culture plates
 p-Aminodimethylaniline oxalate
 Glass slides
 Bunsen burner
 Tooth picks
 Distilled water
 Beaker
 Micropipette
 Micropipette tips (1ml)
 Match box
 Ethanol
 Tissue paper
 Cotton
Methodology:
Filter Paper Method:
1. Filter paper was placed in a petri dish.
2. Sample (test organism) was obtained with the help of sterile tooth pick and gently smeared it on the
filter paper.
3. One or two drops of p-aminodimethylaniline oxalate reagent were placed on the test organism.
4. The test organism was observed for the appearance of a purple color within 30 seconds of contact
with the oxidase reagent, indicating a positive test.
Results:
Purple color was appeared within 30 seconds of contact with the oxidase reagent, indicating a positive
test.

Figure: Oxidase test-Filter paper method