MB Lab Gopal

14/4/2011
COMPREHENSIVE HAND HELD

B 09
PUBLISHERS MICROBIOLOGY LABORATORY
MANUAL
Mr. S. Venkatesh ( Visiting Faculty), AUT-T | Gopalakrishnan C

CONTENTS
1. LABORATORY SAFETY AND STERILIZATION TECHNIQUES
2. ISOLATION AND IDENTIFICATION OF MICROORGANISMS
PREPARATION OF CULTURE MEDIA:
a) Solid media
b) Liquid media
CULTURING OF MICRO ORGANISMS:
a) Pour plate
b) Spread plate
c) Streak plate
3. ISOLATION AND IDENTIFICATION OF MICRO ORGANISMS (SOIL AND MILK)
a) Serial dilution
b) Staining techniques
1. Simple staining
2. Differential staining
c) Bio chemical test
4. QUANTIFICATION OF MICROORGANISMS
5. EFFECT OF DISINFECTANTS ON MICROBIAL FLORA
6. ANTIBIOTIC SENSITIVITY TEST
7. GROWTH CURVE
a) Bacteria
b) Yeast
8. EFFECT OF DIFFERENT PARAMETERS ON BACTERIAL GROWTH CURVE
a) PH
b) Temperature
c) UV irradiation
Microbiology Laboratory Manual – AUT-T Page 2

BT1258 – MICROBIOLOGY LABORATORY
LTPC
0042
LIST OF EXPERIMENTS
1. Laboratory safety and sterilization techniques

2. Microscopic methods in the identification of microorganisms
3. Preparation of culture media – nutrient broth and nutrient agar
4. Culturing of microorganisms in broth and in plates (pour plates, streak plates, isolation
and preservation of bacterial cultures)
5. Staining techniques – Gram's and differential
6. Quantification of microorganisms.
7. Effect of disinfectants on microbial flora
8. Isolation and identification of microorganisms from different sources – soil, water and
milk
9. Antibiotic sensitivity assay
10. Growth curve – Observation and growth characteristics of bacteria and yeast
11. Effect of different parameters on bacterial growth (pH, temperature & UV irradiation)
Total: 60
REFERENCE
1. Micro Biology: Laboratory Theory and applications, M.J. Heboffee aw BE Pierce

Morten Publishing House, 2006.
EQUIPMENTS / APPARATUS
1. Microbiological Hood for sterilization with UV lighting (One).
2. Bunsen Burners – 15 Nos.
3. Orbital Shaker and incubator – 2 Nos.
4. Refrigerator – 1 No.
5. Reagents and consumables – Required amount.
.

«ñ½¡ ¦¾¡Æ¢øÑðÀô Àø¸¨Äì¸Æ¸õ, ¾¢Õîº
¢Ã¡ôÀûÇ¢
¾¢Õîº¢Ã¡ôÀûÇ¢ - 620024
___________________________________________________________________________
Mr. S.Venkatesh M. Phil(Bioinfo)., M. Phil(Biotech)., Ph.D Date:13.04.11
Visiting Faculty, Dept. of Biotechnology
AUT-T
I the author very glad to present this manual inorder to improvise the students in their
laboratory skills and to potentially equip them with the overall knowledge of the various techniques in the
microbiology.
I am sure that this manual will inculcate a passion towards microbiology amongst the
students and will serve as a better reference source for each and every student ever…
S.Venkatesh
Mobile: +91-9894164684 Email: venkatesh.au@gmail.com

MICROBIOLOGY LAB MANUAL
EX:NO:1 MICROBIOLOGY LABORATORY SAFETY
RULES:
1. All materials and clothes other than those needed for the laboratory are to be kept
away from the work area.
2. A lab coat or other protective clothing must be worn during lab. The lab clothing is
not to be worn outside of the laboratory.
3. Clean the lab table before and after lab with the disinfectant solution provided
4. Wash hands before leaving lab.
5. Any item contaminated with bacteria or body fluids must be disposed of properly.
Disposable items are to be placed in the BIOHAZARD container. Reusable items are
to be placed in the designated area for autoclaving prior to cleaning. Sharps are to be
disposed of in the appropriate container
6. Reusable items should have all tape and marks removed by the student before being
autoclaved.
7. Because organisms used in this class are potentially pathogenic, aseptic technique
must be observed at all times. NO eating, drinking, application of cosmetics or
smoking is allowed. Mouth pipetting is not allowed.
8. Cuts and scratches must be covered with Band-Aids. Disposable gloves will be
provided on request.
9. Long hair should be tied back while in lab.
10. All accidents, cuts, and any damaged glassware or equipment should be reported to
the lab instructor immediately.
11. Sterilization techniques will involve the use of Bacticinerators that are fire and burn
hazards. Bacticinerators reach an internal temperature of 850o C or 1500o F. Keep all
combustibles away from the Bacticinerators. Do not leave inoculating loops or
needles propped in the Bacticinerator.
12. Microscopes and other instruments are to be cared for as directed by the instructor.
13. It is the responsibility of the student to know the location and use of all safety
equipment in the lab (eyewash, fire extinguisher, etc.)
14. Cultures may not be removed from the lab. Visitors are not allowed in the lab.
15. Doors and windows are to be kept closed at all times.

EX: NO:4A THE STREAK-PLATE TECHNIQUE
Aim:
1. To understand the purpose of the streak-plate technique.
2. To perform a streak-plate technique and isolate discrete colonies for subculturing
.MATERIALS:
 24- to 48-hour Luria broth cultures of Escherichia coli

 Boiling water bath
 48° to 50°C water bath
 Bunsen burner
 Petri plates
 Inoculating loop
PRINCIPLE:
A mixed culture contains two or more bacterial species that are known and can be
easily separated based on cultural or biochemical characteristics. Streak plates allow for the
growth of isolated colonies on the surface of the agar. An isolated colony is a colony that is
not touching any other colonies and is assumed to be a pure culture. They also show colonial
morphology that may be useful in identifying the organism. Part of the identification of any
organism includes a description of colonial morphology. Other elements of a colonial
description include colony color, hemolysis (if grown on blood agar), form, elevation and
margin.
PROCEDURE:
1. Label the sterile nutrient agar plate with the source of the culture and your initials.
2. Sterilize the loop.
3. Using appropriate aseptic technique, remove a loopful of broth from the mixed culture
tube.
4. Lift the agar plate from the lid and streak about half of the plate. The loop should be
parallel to the agar surface to prevent digging into or gouging the agar.

5.Return the plate to the lid. Sterilize the loop. Lift the agar plate and make one streak into
the inoculated portion of the plate. Finish by streaking about one fourth of the uninoculated
plate.
6. Return the plate to the lid. Sterilize the loop. Lift the agar plate and make one streak into
the second inoculated portion of the plate. Finish by streaking the remaining one-fourth of the
uninoculated plate. Sterilize the loop.
7. Place the plate in a 37o C incubator for 24-48 hours. Observe for growth and record your
results in the table provided at the end of the procedure section.
EX: NO:4B THE POUR-PLATE TECHNIQUE

AIM:
1. To understand the purpose of the pour-plate technique.
2. To perform a pour-plate technique and isolate discrete colonies for subculturing
.MATERIALS:
 24- to 48-hour Luria broth cultures of Escherichia coli

 Boiling water bath
 48° to 50°C water bath
 Bunsen burner
 Petri plates
 Inoculating loop
PRINCIPLE:
The pour plate is used for counting organisms in a solution. A standard volume of
solution is mixed in the liquefied agar. Each organism in the solution is separated from all
others. When the agar solidifies the cells are trapped in the agar and develop into colonies.
Each colony can be counted and represents a single cell in the original solution. If a milliliter
of solution is mixed in the agar then the number of colonies represents the number of
organisms per milliliter of solution. When evaluating a solution for bacteria a series of
dilutions is usually made and cultured. The plate with colonies is counted and the number
multiplied by the dilution factor for that plate to determine the number of bacteria per
milliliter in the original solution. This method is used to evaluate the number of organisms in
milk, drinking water, and even the water at the beach. While the cells grow and are isolated
from each other in a pour plate, they will not develop typical colonial morphology and are not
easily accessible for further testing.
PROCEDURE:
1. Label the bottom of the sterile petri plate with the source of the culture and your
initials. Turn the plate so the lid is facing up.
2. Obtain two tubes of liquefied nutrient agar, one for each student in the pair. The nutrient
agar was boiled (100o C) to melt the agar. Agar at that temperature would kill bacteria, so the
agar has been cooled to 60o C and held in a water bath to maintain that temperature. This

should not kill the bacteria when they are introduced to the liquid agar and will also reduce
the amount of condensation that will collect on the lid of the petri dish.
3. The agar will solidify at 42o C. One student of the pair should aseptically transfer two
drops of the mixed culture broth to one of the agar tubes. The other student should aseptically
transfer one drop of the mixed culture broth the second agar tube. Mix the tubes by rolling the
tubes between your hands, then pour the inoculated liquid agar into a labeled sterile petri
dish. Gently move the dish in a figure eight to completely cover the bottom of the dish with
agar.
4. Allow the agar to solidify. Add to the labeling the amount of mixed culture used in the
agar. A milliliter contains approximately 20 drops. Two drops would be approximately 0.1 ml
and 1 drop would be approximately 0.05 ml. 5. Incubate the plates at 37o C for 24-48 hours.
Examine the plates for growth and record the results in the table below.

EX NO: SPREAD PLATE TECHNIQUE
AIM
1. Understand the purpose of the spread-plate
technique
2. Perform the spread-plate technique
MATERIALS:
• 24- to 48-hour LURIA broth cultures of Bacillus subtilis

• Bunsen burner
• inoculating loop
• 95% ethyl alcohol
• L-shaped glass rod
• Marker pen
• agar plates
PRINCIPLE:
In natural habitats, bacteria usually grow together in populations containing a number
of species. In order to adequately study and characterize an individual bacterial species, one
needs a pure culture. The spreadplate technique is an easy, direct way of achieving this
result. In this technique, a small volume of dilute bacterial mixture containing 100 to 200
cells or less is transferred to the center of an agar plate and is spread evenly over the surface
with a sterile, L-shaped glass rod. The glass rod is normally sterilized by dipping in alcohol
and flamed to burn off the alcohol. After incubation, some of the dispersed cells develop into
isolated colonies. A colony is a large number of bacterial cells on solid medium, which is
visible to the naked eye as a discrete entity. In this procedure, one assumes that a colony is
derived from one cell and therefore represents a clone of a pure culture. After incubation, the
general form of the colony and the shape of the edge or margin can be determined by looking
down at the top of the colony. The nature of the colony elevation is apparent when viewed
from the side as the plate is held at eye level. These variations are illustrated in. After a well-
isolated colony has been identified, it can then be picked up and streaked onto a fresh
medium to obtain a pure culture.
PROCEDURE:
1. With a marker pen, label the bottom of the agar medium plates with the name of the
bacterium to be inoculated, your name, and date. The plates are to be inoculated: E. coli
2. Pipette 0.1 ml of the respective bacterial culture onto the center of a tryptic agar plate
3. Dip the L-shaped glass rod into a beaker of ethanol and then tap the rod on the side of the
beaker to remove any excess ethanol.
4. Briefly pass the ethanol-soaked spreader through the flame to burn off the alcohol, and
allow it to cool inside the lid of a sterile petri plate.
5. Spread the bacterial sample evenly over the agar surface with the sterilized spreader,
making sure the entire surface of the plate has been covered. Also make sure you do not
touch the edge of the plate.
6. Immerse the spreader in ethanol, tap on the side of the beaker to remove any excess
ethanol, and
reflame.
7. Repeat the procedure to inoculate the remaining two plates.
8. Invert the plates and incubate for 24 to 48 hours at room temperature or 30°C.
9. After incubation, measure some representative colonies and carefully observe their
morphology. Record your results in the report.

EX: NO:4C ISOLATION AND PRESERVATION OF
BACTERIAL CULTURES
AIM:
1. Use aseptic technique to remove and transfer bacteria for subculturing
2. Prepare a stock culture using the isolates
MATERIALS:
1. inoculating loop
2. inoculating needle
3. Bunsen burner
4. blow-out pipette with pipettor
5. to-deliver pipette
6. 24-hour broth and agar
7. slant cultures
8. broth tubes
9. agar slants
10. agar deeps
11. marker pen
PRINCIPLE:
Once discrete, well-separated colonies develop on the surface of the streak plate,
selected ones may be picked up with an inoculating needle and transferred to separate culture
tubes, such as agar slants. Where possible, bacteria from the center of a colony are
transferred, because the center is less likely to be contaminated than the edges. Each slant
now represents the growth of a single species of microorganism and is called a pure or stock
culture. One of the more important problems in a microbiology laboratory is the maintenance
of pure stock cultures over a long period. Ideally, one should employ a technique that
minimizes the need for subculturing the microorganism. This is achieved by storing the
microorganism in a state of dormancy either by refrigeration or desiccation. One way in
which many cultures may be maintained for relatively long periods is by sealing them with
sterile mineral oil in order to prevent moisture loss. A number of genera may be stored under
oil (e.g., Bacillus, most Enterobacteriaceae, some species of Micrococcus, Proteus,
Pseudomonas, and Streptococcus). The best way to preserve many stock cultures for long
periods is through lyophilization (freeze-drying). This eliminates the need for periodic
transfers and reduces the chance of mutations occurring in the stock culture. In lyophilization,
the bacterial culture is suspended in a sterile solution of some protective medium such as
milk, serum, or 3% lactose. Small amounts of the thick suspension are transferred to vials and
then quickly frozen in a dry-ice/alcohol mixture. The frozen suspension is finally dried under
vacuum while still frozen, and the vial sealed. These sealed, desiccated cultures may often be
stored for years.
PROCEDURE:
1. With a marker pen, label the agar slants with the names of the respective bacteria. Do the
same for the broth tubes. Add your name and date.
2. Using aseptic technique, select a well-isolated colony for each of the three bacteria and
pick off as much of the center of the colony as possible with an inoculating loop. It may be
necessary to obtain material from more than one colony. Prepare a slant culture and a broth
tube for each of the bacteria. If screw-cap tubes are used, they must be loosened slightly
before incubation to keep the slant aerobic.
3. After incubating 24 to 48 hours, you should have three pure slant and three pure broth
stock cultures.
4. Observe the broth cultures while taking care not to agitate them. Record your observations
in the report.
5. Place the pure cultures in the refrigerator for later use.

Glycerol Stock
EX: NO:5 GRAM’S STAINING

AIM:
1. To understand the biochemistry underlying the Gram stain
2. To understand the theoretical basis for differential staining procedures
3. To differentiate a mixture of bacteria into gram-positive and gram-negative cells
MATERIALS:
1. 18- to 24-hour broth cultures of formalinized, Escherichia coli
2. solutions of crystal violet, Gram’s iodine
3. 95% ethanol and/or isopropanol-acetone mixture (3:1 v/v), and safranin
4. clean glass slides
5. inoculating loop
6. Bunsen burner
7. bibulous paper
8. microscope
9. lens paper and lens cleaner
10. immersion oil
11. slide warmer
12. staining rack
PRINCILPLE:
Simple staining depends on the fact that bacteria differ chemically from their
surroundings and thus can be stained to contrast with their environment. Bacteria also differ
from one another chemically and physically and may react differently to a given staining
procedure. This is the principle of differential staining. Differential staining can distinguish
between types of bacteria. The Gram stain (named after Christian Gram, Danish scientist
and physician, 1853–1938) is the most useful and widely employed differential stain in
bacteriology. It divides bacteria into two groups— gram negative and gram positive. The
first step in the procedure involves staining with the basic dye crystal violet. This is the
primary stain. It is followed by treatment with an iodine solution, which functions as a
mordant; that is, it increases the interaction between the bacterial cell and the dye so that the
dye is more tightly bound or the cell is more strongly stained. The smear is then decolorized
by washing with an agent such as 95% ethanol or isopropanol-acetone. Gram-positive
bacteria retain the crystal violet-iodine complex when washed with the decolorizer, whereas
gram-negative bacteria lose their crystal violet-iodine complex and become colorless. Finally,

the smear is counterstained with a basic dye, different in color than crystal violet. This
counterstain is usually safranin. The safranin will stain the colorless, gram-negative bacteria
pink but does not alter the dark purple color of the gram-positive bacteria. The end result is
that gram-positive bacteria are deep purple in color and gram-negative bacteria are pinkish to
red in color.
PROCEDURE:
1. Prepare heat-fixed smears of E. coli
2. Place the slides on the staining rack.
3. Flood the smears with crystal violet and let stand for 30 seconds.
4. Rinse with water for 5 seconds.
5. Cover with Gram’s iodine mordant and let stand for 1 minute.
7. Decolorize with 95% ethanol for 15 to 30 seconds. Do not decolorize too long. Add the
decolorizer drop by drop until the crystal violet fails to wash from the slide. Alternatively, the
smears may be decolorized for 30 to 60 seconds with a mixture of isopropanol-acetone (3:1
v/v).
9. Counterstain with safranin for about 60 to 80 seconds. Safranin preparations vary
considerably in strength, and different staining times may be required for each batch of stain.
11. Blot dry with bibulous paper and examine under oil immersion. Gram-positive organisms
stain blue to purple; gram-negative organisms stain pink to red. There is no need to place a
coverslip on the stained smear.

IDENTIFICATION OF MICRO ORGANISMS USING BIOCHEMICAL
TESTS
AIM:
Bacteria can be accomplished on the basis of their biochemical properties and enzymatic
reactions in the presence of specific substrates. IMViC is a mnemonic to remember the four
biochemical tests being used: Indole, Methyl red, Voges-Proskauer, and Citrate. These four
tests help divide the Enterobacteriaceae into two major groups—the E. coli group and the
Enterobacter-Klebsiella group.
Principle
Indole
Organisms that posses the enzyme tryptophanase can break down the amino acid tryptophan
to indole. When indole reacts with para-dimethylaminobenzaldehye (Kovac’s reagent) a pink
-colored complex is produced. Tryptophan is plentiful in most media, but growth on blood
agar or chocolate agar produces the best effects.
Procedure:
1. Using sterile technique inoculate each experimental organism into its appropriately
labeled deep tube by means of a stab inoculation. The last tube will serve as a
control.
2. Incubate tubes 24 to 48 hours at 37oC

Methyl Red
Some organisms produce acid from the metabolism of glucose in a sufficient quantity
to produce a pH of 4.4 in the media. These acids are not further metabolized and are said to
be stable acids. At a pH of 4.4 or less the pH indicator methyl red is a bright cherry red.
Procedure:
1. Using sterile technique, inoculate each experimental organism into its appropriately
Labeled tube of media by means of a loop inoculation. The last tube will serve as a
control.
2. Incubate all cultures for 24 to 48 hours at 37oC

Voges-Proskauer
Some organisms initially produce acid from glucose metabolism but further
metabolize the acid produced to neutral end products, such as acetoin, and 2,3-butanediol.
Initially the pH may drop to 4.4 but the neutral end products raise the pH so the methyl red
test will be negative. Acetoin and 2,3 –butanediol under alkaline conditions will react with
alpha-naphthol (1-naphthol) to produce a mahogany red color.
Procedure:
Labeled tube of media by means of a loop inoculation. The last tube will serve as a
control.
2. Incubate all cultures for 24 to 48 hours at 37oC.
Citrate
Citrate contains carbon. If an organism can use citrate as its only source of carbon the
citrate in the media will be metabolized. Bromthymol blue is incorporated into the media as
an indicator. Under alkaline conditions, this indicator turns from green to blue. The utilization
of citrate in the media releases alkaline bicarbonate ions that cause the media pH to increase
above 7.4.
Procedure:
Labeled tube of media by means of a streak inoculation. The last tube will serve as a
control.
2. Incubate all cultures for 24 to 48 hours at 37oC.
EX NO: 06 QUANTIFICATION OF MICROORGANISMS ( BACTERIA)

AIM:
1. To describe several different ways to quantify the number of bacteria in a given sample
2.To determine quantitatively the number of viable cells in a bacterial culture by the standard
plate count technique
3. To measure the turbidity of a culture with a spectrophotometer and relate this to the
number (biomass) of bacteria.
MATERIALS:
1. 24-hour broth culture of Escherichiacoli
2. 4 sterile 99-ml saline or phosphate buffer blanks
3. 1-ml or 1.1-ml pipettes with pipettor
4. 6 petri plates
5. 6 agar pour tubes of yeast agar (plate count agar)
6. 48° to 50°C water bath
7. boiling water bath
8. Bunsen burner
9. cuvettes
10. spectrophotometer
11. 4 tubes of broth
PRINCIPLE:
Many studies require the quantitative determination of bacterial populations. The two
most widely used methods for determining bacterial numbers are the standard, or viable,
plate count method and spectrophotometric (turbidimetric) analysis. Although the two
methods are somewhat similar in the results they yield, there are distinct differences. For
example, the standard plate count method is an indirect measurement of cell density and
reveals information related only to live bacteria. The spectrophotometric analysis is based on
turbidity and indirectly measures all bacteria (cell biomass), dead or alive. The standard plate
count method consists of diluting a sample with sterile saline or phosphate buffer diluent until
the bacteria are dilute enough to count accurately. That is, the final plates in the series should
have between 25 and 250 colonies. Fewer than 25 colonies are not acceptable for statistical
reasons, and more than 250 colonies on a plate are likely to produce colonies too close to
each other to be distinguished as distinct colony-forming units (CFUs). The assumption is
that each viable bacterial cell is separate from all others and will develop into a single

discrete colony (CFU). Thus, the number of colonies should give the number of live bacteria
that can grow under the incubation conditions employed. A wide series of dilutions (e.g., 10–
4 to 10–10) is normally plated because the exact number of live bacteria in the sample is
usually unknown. Greater precision is achieved by plating duplicates or triplicates of each
dilution. Increased turbidity in a culture is another index of bacterial growth and cell numbers
(biomass). By using a spectrophotometer, the amount of transmitted light decreases as the
cell population increases. The transmitted light is converted to electrical energy, and this is
indicated on a galvanometer. The reading indirectly reflects the number of bacteria. This
method is faster than the standard plate count but is limited because sensitivity is restricted to
bacterial suspensions of 107 cells or greater.
PROCEDURE:
STANDARD PLATE COUNT
1. With a marker pen, label the bottom of six petri plates with the following dilutions: 10 –4,
10–5, 10–6, 10–7, 10–8, and 10–9. Label four bottles of saline or phosphate buffer 10–2, 10–4, 10-6,
and 10–8.
2. Using aseptic technique, the initial dilution is made by transferring 1.0 ml of liquid sample
or 1 g of solid material to a 99-ml sterile saline blank. This is a 1/100 or 10–2 dilution. Cap the
bottle.
3. The 10–2 blank is then shaken vigorously 25 times by placing one’s elbow on the bench and
moving the forearm rapidly in an arc from the bench surface and back. This serves to
distribute the bacteria and break up any clumps of bacteria that may be present.
4. Immediately after the 10–2 blank has been shaken, uncap it and aseptically transfer 1.0 ml
to a second 99-ml saline blank. Since this is a 10–2 dilution, this second blank represents a 10–4
dilution of the original sample. Cap the bottle.
5. Shake the 10–4 blank vigorously 25 times and transfer 1.0 ml to the third 99-ml blank. This
third blank represents a 10–6 dilution of the original sample. Cap the bottle. Repeat the
process once more to produce a 10–8 dilution.
6. Shake the 10–4 blank again and aseptically transfer 1.0 ml to one petri plate and 0.1 ml to
another petri plate. Do the same for the 10–6 and the 10–8 blanks.
7. Remove one agar pour tube from the 48° to 50°C water bath. Carefully remove the cover
from the 10–4 petri plate and aseptically pour the agar into it. The agar and sample are
immediately mixed by gently moving the plate in a figure-eight motion while it rests on the
tabletop. Repeat this process for the remaining five plates.

8. After the pour plates have cooled and the agar has hardened, they are inverted and
incubated at 35°C for 24 hours or 20°C for 48 hours.
9. At the end of the incubation period, select all of the petri plates containing between 25 and
250 colonies. Plates with more than 250 colonies cannot be counted and are designated too
numerous to count (TNTC). Plates with fewer than 25 colonies are designated too few to
count (TFTC). Count the colonies on each plate. If at all possible, a special counter such as a
Quebec colony counter should be used. Your instructor will demonstrate how to use this
counter or a handheld counter.
10. Calculate the number of bacteria (CFU) per milliliter or gram of sample by dividing the
number of colonies by the dilution factor. The number of colonies per ml reported should
reflect the precision of the method and should not include more than two significant figures.
For example, suppose the plate of the 10–6 dilution yielded a count of 130 colonies. Then, the
number of bacteria in 1 ml of the original sample can be calculated as follows: Bacteria/ml =
(130) ÷ (10–6) = 1.3 × 108 or 130,000,000.
11.Record your results.
TURBIDIMETRY DETERMINATION OF BACTERIAL NUMBERS

1. Put one empty tube and four tubes of the sterile broth in a test-tube rack. With the
exception of the empty tube, each tube contains 3 ml of sterile broth. Use four of these tubes
(tubes 2 to 5) of broth to make four serial dilutions of the culture.
2. Standardize and use the spectrophotometer as follows:
a. Turn on the spectrophotometer by rotating knob B in to the right.
b. Set the monochromator dial so that the correct wavelength in nanometers (550 to 600 nm)
is lined up with the indicator in the window adjacent to this dial.
c. When there is no cuvette in the cuvette holder, the light source is blocked. The pointer
should thus read zero transmittance or infinite absorbance. This is at the left end of the scale.
Turn knob B until the pointer is aligned with the left end of the scale. d. Place in the cuvette
holder the cuvette that contains just sterile broth. This tube is called the blank because it has
a sample concentration equal to zero. It should therefore have an absorbance of zero (or a
transmittance of 100%). This is at the right end of the scale. Set the pointer to the right end of
the scale using knob C .
e. Place the other cuvettes, which contain the diluted bacterial suspension, in the cuvette
chamber one at a time. Repeat steps c and d between experimental readings to confirm
settings.

f. Close the hatch and read the absorbance values of each bacterial dilution, and record your
values. Remember to mix the bacterial suspension just before reading its absorbance.
g. Record your values. Using the plate count data, calculate the colonyforming units per
milliliter for each dilution.

EX NO: 8A ENUMERATION OF MICROORGANISMS
(SOIL and WATER)
AIM:
1. Become acquainted with some of the microorganisms present in garden soil
2. Determine the number of bacteria, actinomycetes, and fungi in the garden soil using the
plate count method.
MATERIALS:
1. 1 g of rich garden soi
2. Sterile water
3. 12 petri plates
4. 1-ml pipettes with pipettor
5. 1 50-ml flask of melted LB agar
6. 1 50-ml flask of melted PDA agar
7. 1 50-ml flask of melted Actinomycetes agar
9. Bunsen burner
10. Marker pen
PRINCIPLE:
Actinomycetes (including actinoplanetes, nocardioforms, and streptomycetes), other
bacteria, and filamentous fungi (Rhizopus, Mucor, Penicillium, and Aspergillus) are all
important members of the soil microbial community. Each gram of rich garden soil may
contain millions of these micro- and macroorganisms. Since soils vary greatly with respect to
their physical features (e.g., pH, general type, temperature, and other related factors), the
microorganisms present will also vary. For example, acid soils will have a higher number of
fungi compared to alkaline soils, and rich garden soil will contain more actinomycetes than
either the other bacteria or fungi. Not surprisingly, no single technique is available to count
the microbial diversity found in average garden soil. Thus, in this exercise, each group of
students will try to determine only the relative number of fungi, actinomycetes, and other
bacteria in a sample of garden soil using the serial dilution agar plating procedure. To support
the three different groups of microorganisms, you will use three types of media:

(1) Potato dextrose agar for the isolation of fungi,
(2) Actinomycetes agar for the isolation of actinomycetes, and
(3) LB agar for the isolation of other bacteria.
PROCEDURE:
1. The procedure for enumeration of soil microorganisms is illustrated
2. Place 1 g of garden soil in a 99-ml sterile water blank. Mix the soil and water thoroughly
by shaking the water-soil mixture vigorously for 3 minutes, keeping your elbow on the lab
table. Transfer 1 ml of this mixture to the second water blank and mix as above. Transfer 1
ml of this mixture to the third water blank and mix as above.
3. Using a marker pen, label three sets of four petri plates each as follows: actinomycetes (10 –
3, 10–4, 10–5, and 10–6), fungi (10–2, 10–3, 10–4, and 10–5), and other bacteria (10–4, 10–5, 10–6, and
10–7). Be sure to use the correct medium for each type of microorganism.
4. Using a 1-ml pipette and aseptic technique, distribute the proper amount of each soil
dilution to the respective petri plates.
5. Remove the melted actinomycetes agar from the water bath and pour 15 ml into each of the
actinomycetes petri plates. Mix on a flat surface, using a circular motion, and allow to
harden. Do the same for the PDA and LB agars.
6. Invert the petri plates and incubate for 3 to 7 days at room temperature. Observe daily for
the appearance of colonies. Count the plates with fewer than 250 colonies but more than 25.
Designate plates with over 250 colonies as too numerous to count (TNTC) and those with
less than 25 colonies as too few to count (TFTC). Record your data.
7. Determine the number of respective microorganisms per milliliter of original culture (gram
of soil) as follows:
Microorganisms per gram of soil = count per plate/dilution used
MEDIA SERIAL DAYS OF INCUBATION

DILUTI
ON
DA DAY 2 DAY 3
Y1
LURIA 10-3 Bact Actinom Fungus

AGAR eria ycetes
10-4 Bact Actinom Fungus

eria ycetes
10-5 Bact
eria
10-6 Bact
eria
ACTINOMYC 10-3 Actinom Actinomy

ETES AGAR ycetes cetes
10-4
10-5
10-6
POTATO 10-3 Fungus

DEXTROSE
10-4
AGAR
10-5
EX NO: 09 ANTIBIOTIC SENSITIVITY TEST

AIM:
1. Observe and evaluate the effects of antibiotics on gram-negative and gram positive
vegetative bacterial cells, both endospore and non-endospore forming.
2. Define antibiotics (narrow-spectrum and broad-spectrum) and chemotherapeutic
agents.
3. Explain how the majority of antibiotics accomplish selective toxicity
MATERIALS:
1. LB culture of Escherichia coli
2. LB agar plates
3. Sterile cotton-tipped applicators
4. Vials containing antibiotic disks of the following:
a. Ampicillin
b. Gentamycin
c. Chloramphenicol
d. Erythromycin
e. Kanamycin
f. Vanamycin
g. Streptomycin
5. Forceps
6. Alcohol for flaming
7. Bunsen burner
PRINCIPLE:
Chemicals that are used to treat disease are termed chemotherapeutics. Antibiotics are
members of this group of chemicals. Antibiotics were originally produced through bacterial
and fungal metabolic reactions and were named as such because they were found to stop or
inhibit the growth and reproduction of microorganisms. There are two bacterial genera that
produce most antibiotics - Streptomyces and Bacillus. The fungal genus Penicillum is
responsible for many other antibiotics. Those chemotherapeutics that are produced by a
chemical lab, like 75 sulfa drugs, are not considered antibiotics.
Chemotherapeutics and Sensitivity Testing
Ehrlich, in the 1800's, discovered that chemotherapeutics were the only drugs that
displayed selective toxicity and were therefore useable when treating disease in humans.

Antibiotics can becategorized as broad-spectrum which denotes effectiveness against a wide
range of bacteria or narrow-spectrum which indicates effectiveness against very few species.
The Kirby-Bauer Antibiotic Disk Diffusion Test is the most widely used test to
observe the sensitivity of a pathogen to various chemotherapeutic agents. This is a
standardized test that involves comparing the turbidity of a bacterial saline suspension with
that of the McFarland No. 5 turbidity standard and then seeding Mueller-Hinton agar plates
with the bacterial solution. After drying, disks containing a specific amount of antibiotic is
placed on the agar and the plates are then incubated at 35oC for 16-18 hours. After
incubation, zones of inhibition are measured and the diameter is compared to a standardized
table and bacteria are grouped into three categories – ‘R’ Resistant, ‘S’ Sensitive
(susceptible), ‘I’ Inconclusive.
PROCEDURE:
1. Obtain 2 LB agar plates and label appropriately.
2. Spread one organism on each of the two plates specified for that species using the cotton-
tipped applicators.
3. Place 3 antibiotic disks on one plate and other 3 on the other making certain they are well
spaced and that every two plates contains all 6 antibiotics (chemotherapeutics).
4. Place the 2 plates in the incubator at 35oC for 24 hours.
5. Measure the zones of inhibition on each plate and record findings.
6. Using the evaluation table, determine resistant, susceptible, or inconclusive for each
species and each antibiotic.
7. Record the results on the report sheet.
ANTIBIOTIC SENSITIVITY REPORT
ANTIBIOTIC INHIBITION ZONE ‘R’, or ‘S’

E. COLI
Ampicillin
Chloramphenicol
Erythromycin
Kanamycin
Gentamycin
Vanamycin
Streptomycin
EX NO: 10A GROWTH CURVE – OBSERVATION AND GROWTH

CHARACTERISTICS OF BACTERIA
AIM :

1. To become familiar with the population growth dynamics of bacterial cultures.
2. To plot a growth curve and determine the generation time of bacterial cultures.
MATERIALS REQUIRED:
CULTURE:
5-10 hr log phase luria broth culture of E. coli.
MEDIA:
Luria broth, nutrient agar
EQUIPMENTS:
37 ⁰C water bath shaker incubator, Biomate 3S spectrophotometer, test tubes, Erylen Meyer flask,
mechanical pipetting device, Orbitech shaker, petri dish, UV-Visible spectrophotometer.
PRINCIPLE:
Under optimum temperature , pH conditions the cells will reproduce rapidly and the
dynamics of microbial growth can be charted by means of a population growth curve,
which is constructed by plotting the increase in cell number versus time of incubation.
The curve can be used to delineate stages of the growth cycle, measurement of cell
numbers and the rate of growth of a particular organism under standardised conditions as
expressed by its generation time, the time required for a microbial population to double.
THE STAGES OF A TYPICAL GROWTH CURVE:
1. LAG PHASE:
During this stage , the cells are adjusting to their new environment. Although the cells
are increasing in size, there is no cell division and therefore no increase in number.
2. LOG PHASE:
In this phase, there is a rapid exponential increase in population, which doubles

regularly until a maximum number of cells is reached. The time required for the
population to double is the generational time. The length of the Log phase varies
depending on the organisms and the composition of the medium.
3. STATIONARY PHASE:

During this stage, the number of cells undergoing division is equal to the number of
cells that are dying . therefore there Is no further increase in cell number, and the
population is maintained at its maximum level for a period of time
4. DECLINE PHASE:
The micro organism die at a rapid and uniform rate. The decrease in population
closely parallels its increase during the Log phase.
PROCEDURE:
1. The luria broth media is prepared for 100 mL in Erylen Meyer flask.
2. Transfer 10 mL of the media into each of the test tubes.
3. Plug the test tubes with cotton and sterilise in autoclave for 20 minutes at a temperature
of 121⁰ C and a pressure of 15 lbs
4. Transfer 1 mL of the innoculum e.coli puc18 to 6 test tubes except 1 test tube which
can be labelled as control.
5. Keep the test tubes in a cotton stuffed beaker and place it in the electronic shaker at
120rpm .
6. The optical density of the media in the control test tube can be measured by UV-visible
spectrophotometer at 550nm.
7. After 30 minutes of interval the 1st test tube is taken from the shaker and the culture is
transferred to the cuvette under aseptic condition and its optical density is measured
using the UV-visible spectrophotometer and the culture media is discarded .
8. The step 8 is repeated for the next 6 test tubes under a time interval of 30minutes.
9. The readings are tabulated.
10. A plot of graph is drawn by taking ‘time of incubation’ on X-axis Vs ‘optical density’
on Y-axis.
11. A hyperbolic growth curve is obtained providing the delineate stages of bacterial
growth.

TABULATION:
TIME OF INCUBATION(mins) OPTICAL DENSITY AT 550 µm
CONTROL
20
40
60
80
100
120

EX NO: 10B GROWTH CURVE – OBSERVATION AND GROWTH
CHARACTERISTICS OF YEAST
AIM :
1. To become familiar with the population growth dynamics of yeast cultures.
2. To plot a growth curve and determine the generation time of yeast cultures.
MATERIALS REQUIRED:
CULTURE:
5-10 hr log phase luria broth culture of saccharomyces cervesceae or commercial yeast
beeds.
MEDIA:
Luria broth, nutrient agar
EQUIPMENTS:
37 ⁰C water bath shaker incubator,
Biomate 3S spectrophotometer, test tubes, Erylen Meyer flask, mechanical pipetting

device, Orbitech shaker, petri dish, UV-Visible Spectrophotometer.
PRINCIPLE:
Under optimum temperature , pH conditions the cells will reproduce rapidly and the
dynamics of microbial growth can be charted by means of a population growth curve,
which is constructed by plotting the increase in cell number versus time of incubation.
The curve can be used to delineate stages of the growth cycle, measurement of cell
numbers and the rate of growth of a particular organism under standardised conditions as
expressed by its generation time, the time required for a microbial population to double.
THE STAGES OF A TYPICAL GROWTH CURVE:

1. LAG PHASE:
During this stage , the cells are adjusting to their new environment. Although the cells
are increasing in size, there is no cell division and therefore no increase in number.
2. LOG PHASE:
In this phase, there is a rapid exponential increase in population, which doubles

regularly until a maximum number of cells is reached. The time required for the
population to double is the generational time. The length of the Log phase varies
depending on the organisms and the composition of the medium.
3. STATIONARY PHASE:
During this stage, the number of cells undergoing division is equal to the number of
cells that are dying . therefore there Is no further increase in cell number, and the
population is maintained at its maximum level for a period of time
4. DECLINE PHASE:
The micro organism die at a rapid and uniform rate. The decrease in population
closely parallels its increase during the Log phase.
PROCEDURE:
1. The luria broth media is prepared for 100 mL in Erylen Meyer flask.
2. Transfer 10 mL of the media into each of the test tubes.
3. Plug the test tubes with cotton and sterilise in autoclave for 20 minutes at a temperature
of 121⁰ C and a pressure of 15 lbs
4. Transfer 1 mL of the innoculum saccharomycer cerevesceae puc18 to 6 test tubes

except 1 test tube which can be labelled as control.
5. Keep the test tubes in a cotton stuffed beaker and place it in the electronic shaker at
120rpm .
6. The optical density of the media in the control test tube can be measured by UV-visible
spectrophotometer at 600 nm.
7. After 30 minutes of interval the 1st test tube is taken from the shaker and the culture is
transferred to the cuvette under aseptic condition and its optical density is measured
using the UV-visible spectrophotometer and the culture media is discarded .
8. The step 8 is repeated for the next 6 test tubes under a time interval of 30 minutes.
9. The readings are tabulated.
10. A plot of graph is drawn by taking ‘time of incubation’ on X-axis Vs ‘optical density’
on Y-axis.

11. A hyperbolic growth curve is obtained providing the delineate stages of yeast growth.
TABULATION:
TIME OF INCUBATION(mins) OPTICAL DENSITY AT 550 µm

CONTROL
30
60
90
120
150
180

EX NO : 11A EFFECT OF TEMPERATURE ON BACTERIA
AIM:
1. Understand how microorganisms are affected by the temperature of their environment
2. Classify these same bacteria based on their temperature preference for growth
3. Determine the effects of heat on bacteria
MATERIALS:
• 24- to 48-hour luria broth cultures of Escherichia coli

• Bunsen burner
• inoculating loop
• refrigerator set at 4°C
• marker
• 48° to 50°C water bath
PRINCIPLE:
Each microbial species requires a temperature growth range that is determined by the
heat sensitivity of its particular enzymes, membranes, ribosomes, and other components. As a
consequence, microbial growth has a fairly characteristic temperature dependence with
distinct cardinal temperatures—minimum, maximum, and optimum. Minimum growth
temperature is the lowest temperature at which growth will occur; maximum growth
temperature is the highest temperature at which growth will occur; and optimum growth
temperature is the temperature at which the rate of cellular reproduction is most rapid. The
optimum temperaturefor the growth of a given microorganism is correlated with the
temperature of the normal habitat of the microorganism.
Most bacteria can be classified into one of three major groups based on their
temperature requirements. Psychrophiles can grow at 0°C and have an optimum growth
temperature of 15°C or lower; the maximum is around 20°C. Mesophiles have growth
optima between 20° and 45°C. The majority of bacteria fall into this category. Thermophiles
can grow at temperatures of 55°C or higher.
Boiling is probably one of the easiest methods of ridding materials of harmful
bacteria. However, not all bacteria are equally sensitive to this high temperature. Some

bacteria may be able to survive boiling even though they are unable to grow. These bacteria
are termed thermoduric. Many of the spore formers (such as B. subtilis) can withstand
boiling for 15 minutes because of their resistant endospores. Thus, both temperature and the
species of bacteria will affect the disinfection of certain specimens. This is important to know
when trying to kill pathogenic bacteria with heat.
PROCEDURE:
1. The plates, glasswares required for experiment was sterilized. The media required for
experimenting with temperature variations was prepared using agar and was
sterilized.
2. Label each section with the name of the test organism to be inoculated. When
labeling the cover of each plate, include the temperature of incubation,(4oC,10oC,37oC
or 60oC).
3. Aseptically inoculate the plates with E.Coli.
4. Incubate all plates in an inverted position at the specified temperatures viz (4oC, 10oC,
37oC or 60oC) for 24 to 38 hours.
5. The plates were taken and results were observed.

EX NO : 11B EFFECT OF pH IN BACTERIA
AIM
1. Understand how pH affects the growth of bacteria

2. Perform an experiment that relates bacterial growth to pH
MATERIALS:
1. 24-hour brothcultures of Escherichia coli
2. pH meter or pH paper
3. sterile petri plates
5. Bunsen burner
6. Petri plates
7. Inoculating loop
Note: The pH is adjusted with either 1 N sodium hydroxide or 1 N acetic acid
PRINCIPLE:
It is not surprising that pH dramatically affects bacterial growth. The pH affects the
activity of enzymes—especially those that are involved in biosynthesis and growth. Each
microbial species possesses a definite Ph growth range and a distinct pH growth optimum.
Acidophiles have a growth optimum between pH 0.0 and 5.5; neutrophiles between 5.5 and
8.0; and alkalophiles 8.5 to 11.5. In general, different microbial groups have characteristic
pH optima. The majority of bacteria and protozoa are neutrophiles. Most molds and yeasts
occupy slightly acidic environments in the pH range of 4 to 6; algae also seem to favor
acidity.
Many bacteria produce metabolic acids that may lower the pH and inhibit their growth. To
prevent this, buffers that produce a pH equilibrium are added to culture media to neutralize
these acids. For example, the peptones in complex media act as buffers. Phosphate salts are
often addedas buffers in chemically defined media. In this exercise, you will work in groups
to see how the pH affects the growth of several microorganisms.

PROCEDURE:
1. Plates and glasswares were sterilised first the media required for showing a variation
in pH was prepared using acid, base over the agar.
2. Label all the four plates with pH , name of the organism on the cover of the plate. The
pH variation shown were 4.5 , 5.5, 6.5 and 7.5
3. Asceptically inoculate all the plate with E.coli.
4. Incubate all the plates in an inverted position for 28 – 48 hours.
5. The plates were taken and the results were observed.

EX: NO: 11C EFFECT OF UV RADIATION ON BACTERIAL
GROWTH:
AIM:
To become acquainted with the microbicidal effect of ultra violet radiation on organisms.
PRINCIPLE:
Certain electromagnetic radiation like gamma radiation, uv radiation which posses
sufficient energy to be microbicidal in nature .these radiations have wavelength lesser than
300nm.ultraviolet light has a lower energy content than many other ionizing radiations. It is
capable of producing a lethal effect in cells exposed to the low penetrating wavelengths in the
range of 210 nm to 300nm cellular components capable of absorbing ultraviolet light or the
nucleic acids with the DNA acting as the primary site of damage. As the pyrimidines
especially absorb ultraviolet wavelengths, the major effect of this form of radiation is
thymine dimerization which is the covalent bonding of the two adjacent thymine molecules
on one nucleic acid strand in the DNA molecule. This dimer formation distorts the
configuration of the DNA molecule. This dimer formation distorts the configuration of the
DNA molecule and the distortion infers with DNA replication and transcription during
protein synthesis. Ultraviolet radiation because of its low penetrability, cannot be used as a
means of sterilization and its practical application is only for surface or air disinfection.
MATERIAL:
Cultures:
24- to 48 hour’s nutrient broth cultures of Escherichia coli.
Media:
LB Agarb
Equipments:
Bunsen buner, inoculating loop, ultraviolet radiation source(254 nm) and glass ware marking
pencil.
PROCEDURE:
1. The plates, glasswares required for experiment was sterilized. The media required for
experimenting with uv light was prepared using agar and was sterilized.
2. Label each of the 4 plates with the name of the test organism to be inoculated and
with the different time interval to which plates are to be subjected with uv light.

3. The plates were exposed to uv light for the specified time interval viz. 10 mins,20
mins,30 mins, 40 mins and the control is maintained at 0 mins to uv exposure.
4. Incubate all plates in an inverted position for 24 to 38 hours.
The plates were taken and results were observed

MB Lab Gopal

Enviado por

Dados do documento

Descrição original:

Título original

Direitos autorais

Formatos disponíveis

Compartilhar este documento

Compartilhar ou incorporar documento

Opções de compartilhamento

Você considera este documento útil?

Este conteúdo é inapropriado?

Direitos autorais:

Formatos disponíveis

MB Lab Gopal

Enviado por

Direitos autorais:

Formatos disponíveis

14/4/2011

COMPREHENSIVE HAND HELD

Mr. S. Venkatesh ( Visiting Faculty), AUT-T | Gopalakrishnan C

1. LABORATORY SAFETY AND STERILIZATION TECHNIQUES

2. ISOLATION AND IDENTIFICATION OF MICROORGANISMS

PREPARATION OF CULTURE MEDIA:

5. EFFECT OF DISINFECTANTS ON MICROBIAL FLORA

6. ANTIBIOTIC SENSITIVITY TEST

Microbiology Laboratory Manual – AUT-T Page 2

1. Laboratory safety and sterilization techniques

1. Micro Biology: Laboratory Theory and applications, M.J. Heboffee aw BE Pierce

Microbiology Laboratory Manual – AUT-T Page 3

Mobile: +91-9894164684 Email: venkatesh.au@gmail.com

Microbiology Laboratory Manual – AUT-T Page 4

4. Wash hands before leaving lab.

9. Long hair should be tied back while in lab.

15. Doors and windows are to be kept closed at all times.

Microbiology Laboratory Manual – AUT-T Page 5

1. To understand the purpose of the streak-plate technique.

2. To perform a streak-plate technique and isolate discrete colonies for subculturing

 24- to 48-hour Luria broth cultures of Escherichia coli

2. Sterilize the loop.

Microbiology Laboratory Manual – AUT-T Page 6

EX: NO:4B THE POUR-PLATE TECHNIQUE

1. To understand the purpose of the pour-plate technique.

2. To perform a pour-plate technique and isolate discrete colonies for subculturing

 24- to 48-hour Luria broth cultures of Escherichia coli

initials. Turn the plate so the lid is facing up.

Microbiology Laboratory Manual – AUT-T Page 8

Microbiology Laboratory Manual – AUT-T Page 9

• 24- to 48-hour LURIA broth cultures of Bacillus subtilis

Microbiology Laboratory Manual – AUT-T Page 11

Microbiology Laboratory Manual – AUT-T Page 13

EX: NO:5 GRAM’S STAINING

Microbiology Laboratory Manual – AUT-T Page 16

Microbiology Laboratory Manual – AUT-T Page 17

2. Incubate tubes 24 to 48 hours at 37oC

Microbiology Laboratory Manual – AUT-T Page 19

EX NO: 06 QUANTIFICATION OF MICROORGANISMS ( BACTERIA)

Microbiology Laboratory Manual – AUT-T Page 20

Microbiology Laboratory Manual – AUT-T Page 21

Microbiology Laboratory Manual – AUT-T Page 22

TURBIDIMETRY DETERMINATION OF BACTERIAL NUMBERS

Microbiology Laboratory Manual – AUT-T Page 23

Microbiology Laboratory Manual – AUT-T Page 24

Microbiology Laboratory Manual – AUT-T Page 26

Microorganisms per gram of soil = count per plate/dilution used

MEDIA SERIAL DAYS OF INCUBATION

LURIA 10-3 Bact Actinom Fungus

10-4 Bact Actinom Fungus

ACTINOMYC 10-3 Actinom Actinomy

POTATO 10-3 Fungus

EX NO: 09 ANTIBIOTIC SENSITIVITY TEST

Microbiology Laboratory Manual – AUT-T Page 29

ANTIBIOTIC SENSITIVITY REPORT

ANTIBIOTIC INHIBITION ZONE ‘R’, or ‘S’

Microbiology Laboratory Manual – AUT-T Page 30

EX NO: 10A GROWTH CURVE – OBSERVATION AND GROWTH

Microbiology Laboratory Manual – AUT-T Page 31

5-10 hr log phase luria broth culture of E. coli.

Luria broth, nutrient agar

THE STAGES OF A TYPICAL GROWTH CURVE: