Mb Lab Gopal

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14/4/2011

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

B 09

PUBLISHERS

COMPREHENSIVE HAND HELD

MICROBIOLOGY LABORATORY

MANUAL



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) Yeast8. 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

L T P C

0 0 4 2

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.

.




___________________________________________________________________________

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: [email protected]


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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 bedisposed 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 theuninoculated 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 thetubes 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

AIM1. Understand the purpose of the spread-platetechnique2. 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. coli2. 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 OFBACTERIAL CULTURESAIM:

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 withalpha-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 cuvetteholder 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

8. 48° to 50°C water bath

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

DILUTI

ON

DAYS OF INCUBATION

DA

Y 1

DAY 2 DAY 3

LURIA 10-3 Bact Actinom Fungus




AGAR eria ycetes

10-4 Bact

eria

Actinom

ycetes

Fungus

10-5 Bact

eria

10-6 Bact

eria

ACTINOMYC

ETES AGAR

10-3 Actinom

ycetes

Actinomy

cetes

10-4

10-5

10-6

POTATO

DEXTROSE

AGAR

10-3 Fungus

10-4

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 C water bath shaker incubator, Biomate 3S spectrophotometer, test tubes, Erylen Meyer flask,⁰

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 populationclosely 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 121 C and a pressure of 15 lbs⁰

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

4060

80

100

120






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 121 C and a pressure of 15 lbs⁰

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,(4 oC,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 plates4. 48° to 50°C water bath

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 isthymine 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

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