AM 1207 Lab Manual

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EXPERIMENT # 01

IDENTIFICATION OF UNKNOWN BACTERIALCULTURES

PURPOSEBy performing this experiment, you will be able to learn the following.

Procedures for the study of morphological, cultural, and biochemical characteristics ofbacterial cultures.

Identification of unknown bacterial cultures on the basis of their characters.GENERAL CONSIDERATIONS

In order to study and identify a microorganism, we must have a pure culture that is a culture

having only one single species of microorganism. The complete systemic collection ofinformation about one species is known as pure culture study. Routine pure culture studyincludes morphological, cultural, biochemical and physiological properties. At this stage you will be

fluent in carrying out methods to study bacterial morphology. Now you should get the knowledge

of how to describe the differing patterns of growth in solid media and broths. You should also beable to study various biochemical and physiological properties of bacteria.

CULTURAL CHARACTERISTICS

The cultural properties of bacterial culture are studied by streak-plate method and also by

inoculating the culture in broth. The streak-plate method is the most popular method for qualitative

isolation of bacteria. A loopful of culture is spread over the surface of an agar medium plate, e.g.

nutrient agar. Although many types of procedures are performed, the four-way streak is usuallyperformed as illustrated in Figure 1. Some bacteria have characteristic growth patterns. Only if these

growth patterns are distinctive for a single species can they aid in the identification of that species.

Although some bacteria do grow in distinctive patterns, many others look very much alike. However,

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you can use these many growth patterns to categorize bacteria. Two types of growth patterns are

classically used in the laboratory.

An isolated colony on the surface of a nutrient agar plate. A nutrient broth culture.COLONIAL CHARACTERISTICSBegin now to study the morphology of an isolated colony by examining Figure 2. The following aspects areused to study the characteristic growth features of a single colony.

Figure 2: Bacterial colonies on an agar plate.

The form: Refer Figure 2 for the names. The size: Pin-pointed --small and large. The margin: Refer Figure 2 for the names of various types of margins. The surface: Smooth/rough/ granular/ringed/ papinate/ glistening. The elevation: Refer Figure 2 for various patterns of elevation. The opacity: Transparent/ translucent/opaque The chromogenesis: The color of the colony such as golden yellow, white, red, etc.

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GROWTH PATTERNS IN BROTH

The growth patterns in nutrient broth (Fig. 3) can be studied by using the following aspects.

Turbidity: Present or absent; uniform, granular or flocculent. Sediments: Present or absent; powdery, granular, floccular, membranous or viscid. Surface growth: Refer Figure 3 for names of different patterns.

GROWTH IN DIFFERENTIAL MEDIA

You will use differential media for the purpose of studying cultural properties of a given bacterial

culture in the laboratory. A differential medium will cause certain colonies to develop differently

(differentiated) from other organisms present.

The differential media arc of great significance in differentiating one group of bacteria from the

other. For example, lactose-fermenting enteric bacteria can be differentiated from lactose-nonfermenters on MacConkey's agar. The former group produces red colonies while later group

produces colorless colonies on this medium. Several other differential media are now-a-days in

use in microbiological labs. You will, however, employ a few of them, such as, Eosin Methyleneblue agar, Brilliant green agar, Triple Sugers Iron agar, Bismith sulfite agar, and mood agar. While

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using such media, you are supposed to record only the differential character of the culture. For

example, in MacConkey's agar it is only the color of the colony which is specially emphasizedand noted.

BIOCHEMICAL CHARACTERISTICS

As mentioned above, many bacteria produce colonies which appear very much alike. Thus, for

their indentification some additional informations are needed. The following biochemical tests

may be of great importance in making a final identification of a bacterium.

IMVIC TESTS

These important diagnostic tests have already been described in preceding exercise.

NITRATE REDUCTION

This test has also been discussed elsewhere.

HYDROGEN SULFIDE PRODUCTION

Some bacteria are able to produce hydrogen sulfide as a result of catabolism of sulfur

containing amino acid (cystine/methionine). The colorless gas, hydrogen sulfide, reacts with anindicator, ferrous ammonium sulfate, to produce an insoluble black precipitate, ferrous sulfide.

These precipitates appear in the butt of Triple Sugar Iron medium commonly employed for this

purpose.

CATALASE PRODUCTION

A complete description of this test has been given elsewhere in this Manual.

COAGULASE PRODUCTIONThe coagulase enzyme is produced by some bacteria which causes coagulation of blood plasma.When a heavy homogenous suspension of such bacteria is mixed with plasma, clumping will take

place in less than 10 seconds.

CARBOHYDRATE FERMENTATION

One of the most important metabolic activities of microorganisms is catabolism ofcarbohydrates. Through these integrated enzymatic reactions, the cells obtain energy and

produce simpler carbon compounds. The bacteria catabolize carbohydrate either by oxidativepathways or fermentative pathways. During oxidative pathways molecular oxygen or inorganicions, such as NO3 or SO4, serve as the final electron acceptor. On the other hand, fermentative

pathways involve the use of organic compounds serve as the final electron acceptor.

Carbohydrate fermentation is basically dependent upon the genetic potential of a bacterium toproduce specific enzymes for the catabolism of a particular carbohydrate. These enzymes cause

dissimilation of the carbohydrate leading to the production of acids, alcohols, and sometimes

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gases such as hydrogen or carbon dioxide.

The enzymatic degradation of carbohydrate through fermentation can be studied by inoculatingbacterial cultures in a fermentation broth. A typical fermentation broth contains basic nutrients,

a specified carbohydrate, and a pH Andrade indicator. The broth tube, most often of 13x100

mm size, also contains a Durham tube, a small, inverted vial that collects gas.

Bacteria that grow in the fermentation broth may ferment the carbohydrate and produce acid or

acid and gas. The production of acid is indicated by the appearance of red or pink color. The gasformation is indicated by the accumulation of gas bubble at the top of Durham tube.

MATERIAL:

CULTURES

24-hour bacterial cultures on agar slopes to be studied and identified.

MEDIA

Used for Gram-negative and Grampositive bacteria. Nutrient agar, Nutrient broth, glucose broth,

lactose broth, sucrose broth, mannitol broth and nitrate broth.

Used for Gram-negative enterics: MacConkey agar, EMB agar, Brilliant green agar, Clark's broth Tryptonebroth, Simmon citrate agar, and TSI agar.

Used for Gram- positive bacilli. Blood Agar.Used for Gram-positive staphylococci. Blood agar, and Mannitol salt agar.

PROCEDURE:

FIRST DAY SESSION

1. A Stain the given cultures by Gram's Method. Check the purity and determine the morphologyarrangement, and Gram reaction of the cells. Record the results in the chart.

2. Using aseptic technique, inoculate the culture in various media.3. Incubate all the sets at 37C for 24 hours.

SECOND DAY SESSION

1. Observe the colonial morphology on Nutrient agar. Determine and record the shape, size,margin, surface, elevation, opacity and color of the colonies. Determine the growth

patterns in the nutrient broth and deferential reaction in various media and record them inthe chart.

2. Perform various biochemical tests and record the results in the chart.3. Identify the organism on the basis of their recorded characters.4. Write briefly the reasons for identification.

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POST TEST

1. What are the three basis of identification of an unknown organism?a. _________________________________

b. _________________________________

c. _________________________________

2. Name the pH indicator used in carbohydrate fermentation broth.___________________________________

3. Fill in the following table.

MEDIUM TYPE INHIBITOR INDICATOR BACTERIA GROWN

MacConkey

EMB

Brilliant green

COMMENTS:

____________________________________________________________________________________

____________________________________________________________________________________

____________________________________________________________________________________

___________________________________________________________________________________

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EXPERIMENT # 02

Isolation of Pure Cultures from a Mix Culture

Basic Concepts

In natural habitats, microorganisms usually in complex, mixed populations containing several species

known as a mixed culture. To study single type of organism, it should be first separated or isolated from

the mixed culture. Such type of isolated culture, which contains viable population of only one of type

organism, is known as pure culture. Axenic culture is a pure culture that has developed from a single

cell.

Development of pure culture technique by Robert Koch transformed microbiology into a much

developed and important science. Within about twenty years most pathogens responsible for the major

human diseases had been isolated of pure cultures is an important job in all fields of microbiology likediagnoses of diseases, taxonomy, immunology, genetics and clinical microbiology.

There are several ways to prepare pure cultures; a few of them are discussed below.

Streak-Plate MethodIn laboratories microorganisms are grown on nutrient preparations called culture media (singular

medium). If a mixture of cells is spread out an agar surface so that every cells grows into a visible cluster

of microorganisms, known as a colony, each colony represents a pure culture. The streak-plate method

is the most popular method for isolation of bacteria. The inoculum is transferred to the edge of an agar

plate with an inoculating loop or swab and then streaked in a four-way pattern as illustrated in Figure

13.1.

Spread-plate MethodThe spread-plate method is an easy, direct way of achieving pure culture. A small volume of dilute

microbial mixture is transferred to the center of an agar plate and spread evenly over the surface with a

sterile bent-glass rod. The individual cells develop into isolated colonies. Spread-plates can be used to

count the microbial population.

Pour-Plate MethodIn pour-plate method, the original sample is diluted several times to reduce the microbial population to

obtain isolated colonies. Small volumes of diluted samples are mixed with melted, called agar medium,

and then poured into sterile petri plates. The medium is allowed to solidify and then incubated. Each cell

is fixed in place and forms an individual colony. each colony represents one viable cell. These colonies

can also be used to prepare pure cultures.

NeedsCulture: Samples of mix culture.

Medium: Nutrient agar plates and nutrient agar slopes.

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Reagents: Saline and Gram stain reagents.

Equipment:Compound microscope, wire loop, glass slides, and Bunsen burner.

Procedure

1. Perform the Gram staining of the given sample of mix culture. Note the different morphologicaltypes of bacteria.

2. Streak the mix culture on the agar plate and incubate at 37o C for 24 hours.3. Select at least two different colonies on the plate and describe their charaters.4. Transfer the marked colonies on respective agar slopes. Incubate at 37o C for 24 hours.5. Gram-stain the isolated cultures on the slopes and check the purity.Test Yourself

1. Name the scientist who has major contributions in the development of pure culture._________________________________________________________________________________

2. Name three methods to isolate pure culture.__________________________________________________________________________________

__________________________________________________________________________________

3. What is the significance of isolated in pure culture?__________________________________________________________________________________

__________________________________________________________________________________

__________________________________________________________________________________

__________________________________________________________________________________

__________________________________________________________________________________

Observations

Cellular Morphology of Bacteria in Mix Culture

Colony code Shape Arrangement Gram reaction

1

23

4

5

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Colonial Morphology of Marked Colonies on Nutrient Agar

Colony Code Colonial Characters

Cellular Morphology of Bacteria in Marked Colonies

Colony Code Shape Arrangement Gram reaction Motility

Comments

_____________________________________________________________________________________

_____________________________________________________________________________________

_____________________________________________________________________________________

_____________________________________________________________________________________

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EXPERIMENT # 03

Escherichia coli

Basic Concepts

The genus Escherichia is named after Theodor Escherich who first isolated these bacteria in 1886. The

genus is a member of tribe Escherichieae. The type specie E.coli is widely distributed in nature. They are

universally found in the intestinal tract of man and both warm and cold-blooded animals. They are

regularly excreted in the faceces. For this reason they are often used as indicators of faecal pollution of

water supplies. E.coli has been one of the most important tools of the microbial geneticist and microbial

physiologist.

Morphology

Like all members of the Enterobacteriaceae they are Gram-negative short rods, motile by peritrichous

flagella. Many strains are encapsulated. E. coli are usually fimbriate possessing both sex piliand adhesivefimbriae.

Cultural Characters

Colonies on nutrient agar are small translucent and colorless. When grown on EMB agar the colonies

typical green metallic sheen. On other differential media produce lactose-fermenting colonies. Some

varieties are beta () haemolytic on blood agar.

Biochemical Reactions

Typical Escherichieae are easily differentiated from other members of the Enterobacteriaceae by their

rapid fermentation of lactose with acid and gas and the classical IMViC tests (++--). E.coliferment most ofsugars with acid and gas. They are also recognized by their typical reactions on TST agar slope acidic,

butt acidic with lot of gas and no hydrogen sulfide.

Pathogenicity

E.coli is quite a notorious bacterium that can cause a variety of infectious diseases involving virtually

every human tissue and organ system. The virulence of E. coli is due to many virulence factors, which

include adhesins, toxins and aggressins.

Urinary tract infections. E. coli is one of the common organisms in urinary tract infection. Such ascystitis, pyelonephiritis, and kidney cortex infections.

Diarrhoealsyndroes. There are four strains of E. coli recognized to be involved in diarrhoealsyndromes. Their disease producing capability is plasmid-mediated.

Enterpathogenic E. colicauses infantile diarrhea. Enteroxigenic E. coli synthesizes heat labile and heat stable enterotoxins that produce secretory

diarrhea (travellers diarrhea).

Enteroinvasive E. colistrains are capable of invading the epithelium of large intestine and producinginflammatory diarrhea.

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Hemorrhagic E.coliforms a powerful cytotoxin that is responsible for hemorrhagic colitis an bloodydiarrhea.

Other infections caused by E. coli include: neonatal meningitis, purpeural fever, and nosocomial

infections.

Test Yourself

1. Name the various virulent strains ofE.coli._____________________________________

_____________________________________

_____________________________________

_____________________________________

2. Name a few diseases caused by virulent E.coli._____________________________________

_____________________________________

_____________________________________

_____________________________________

3. Differentiated between E. coli and S. typhi on the basis of TSI reactions.________________________________________________________________________

____________________________________

____________________________________

____________________________________

____________________________________

____________________________________

____________________________________

____________________________________

____________________________________

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Observations

Morphological characteristics

Culture Code Shape Arrangements Gram reaction

Cultural Characteristics

Culture Code Colonial Morphology

(Nutrient agar)

Growth Pattern

(Nutrient broth)

Differential Colonial Characteristics

Culture Code

Culture Media

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Biochemical Characteristics

Culture Code IMViC Tests

G S L M Ma I MR VP C

TSI agar NO3 Ca Ur Ox Ge*

Butt Slope H2S Gas

Comments

_____________________________________________________________________________________

_____________________________________________________________________________________

_____________________________________________________________________________________

_____________________________________________________________________________________

_____________________________________________________________________________________

_____________________________________________________________________________________

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_____________________________________________________________________________________

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EXPERIMENT # 04

PHOSPHATASE TEST

PURPOSE

This exercise is designed to achieve the following.

Detection of phosphatase enzyme in milk sample. Evaluation of quality of pasteurized milk.

GENERAL CONSIDERATIONS

Pasteurization is defined as a process of heating a liquid food at a controlled temperature todestroy harmful microorganisms and thus to enhance the shelf life. It is a method of disinfection

designed originally by Louis Pasteur. Two methods of pasteurization are commonly employed.

The low -temperature holding (LTH) method exposes milk to 145F (62.8C) for 30 minutes

in special equipment. The high temperature short time (HTST) method employs a temperature of

161F (71.7C) for 15 seconds. In both the methods the equipment is so designed that it heatsevery particle of the liquid food to a specified temperature for the required time. The heating

process is followed by immediate cooling of the pasteurized product.

Phosphatase is a thermolabile enzyme normally present in raw milk. This enzyme is

completely destroyed by pasteurization. The phosphatase test is an important test which is routinelyperformed to determine the quality of pasteurized milk. The presence of phosphatase indicates

inadequate pasteurization, staleness, or illegal adultration with raw milk. The principle of this test

is based on the liberation of phenol by phosphatase from disodium orthophenyl phosphate, thesubstrate of the enzyme.

MATERIALS:

SAMPLES

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Two or more milk samples.

SUBSTRATE

Buffered solution of disodium orthophenyl phosphate.

INDICATOR

2, 6-Dichloroquinonechloroimide (CQC) or ferric chloride, solution.

EQUIPMENTSterile 1-ml and 5-ml pipettes, test tubes, and a water bath.

PROCEDURE

1. Take 5.0 ml of buffered solution of substrate in a test tube and add 0.5 ml of milk sample.2. Label the tube and keep it in a water bath, set at 40C, for 15 minutes.3. Transfer 1.0 ml of the tube contents into another tube and pour 5-6 drops of the indicator.4. Appearance of blue color indicates a positive test.

OBSERVATIONS AND RESULTS

MILK SAMPLE APPEARANCE

OF COLOR

PHOSPHATASE STATUS

OFMILKPRESENT ABSENT

RESULT:-

______________________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________

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POST TEST

TRUE OR FALSE

1. Phosphatase is found only in pasteurize d milk.

2. Phosphatase test is performed to determine the quality of pasteurization.3. The principle of phosphatase test is based on the liberation of phenol by phosphatase.

4. CQC solution is used as a substrate for phosphatase.

5. positive phosphatase test indicates the presence of high number of bacteria.

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EXPERIMENT # 05

IMVIC TESTS

PURPOSE

The IMVIC tests, in fact, are a set of biochemical reactions commonly employed in theidentification of bacterial species, particularly the members of Enterobacteriaceae. Family

Enterobacteriaceae consists of numerous species which are short, Gram-negative, non-sporing

bacilli. Enterobacteriaceae, one of the largest family, has been assigned a lot of significance sinceit includes several pathogens (e.g., species of Salmonella and Shigella), occasional pathogens

(e.g., species ofKlebsiella and Proteus), and members of normal intestinal flora (e.g., species of

Escherichia and Enterobacter). The members of Enterobacteriaceae are usually designated by a

common name, the enterics.

The identification of the enterics, and also many non-enterics, can be accomplished on the basis oftheir biochemical. characteristics. These characteristics, in turn, are determined by the enzyrnaticpotential of the organisms. Besides several biochemical tests, the IMVIC tests can be used in thecharacterization and differentiation of bacterial species.

The acronym "IMViC" is a mnemonic aid for memorizing the order and names of four tests asshown in the following.

I = Indole

M = Methyl redVi = Voges-Proskauer (The "i" is for ease of pronunciation)C = Citrate,

INDOLE TEST

PURPOSE

Indole test is performed in order to learn the following.

Demonstration of the ability or inability of the organisms to produce enzymetyptophanase.

Production of indole as a result of enzymatic degradation of amino acid tryptophan. Application of the test in identifying bacterial species.


Indole is a nitrogenous compound formed as a result of bacterial breakdown of amino acid tryptophan.

This breakdown requires the production of enzyme tryptophanase by certain bacteria. The enzyme

tryptophanase causes hydrolysis of tryptophan and produce metabolic products including indole, skatole,

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and indoleacetic acid. The major intermediate product in the tryptophan degradation is indolepyruvic acid

from which indole is formed through deamination. A summerized reaction is illustrated in the following.

Trytophanase

Tr to hane Indole ruvic

Indolepyruvic acidDeamination

Indole + Pyruvic acid + Ammonia

The medium employed in the indole test is a broth consisting of tryptone dissolved in distilled water. Tryptoneis a pancreatic digest of the milk protein casein and serves as a rich source of amino acid tryptophan. Theproduction of indole is detectable by adding Kovac's reagent in the inoculated broth. Indole, if present,

combines with p-dimethylaminobenzaldehyde in Kovac's reagent and produces quinoidal. This compoundaccumulates in the form of a cherry-red layer. The presence of hydrochloric acid and amyl alcohol is essentialfor the formation of quinoidal.

MATERIAL:

CULTURES

24-hour cultures ofE. coli, Ent. aerogenes,and S. typhi.

MEDIATryptone broth, one tube each for the given cultures.

REAGENT

Kovac's reagent.

EQUIPMENTBunsen burner, 100x13 mm test tubes, and wire loop.

PROCEDURE:

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FIRST DAY SESSION

1. Inoculate the tryptone broth tubes with the given cultures separately.2. Incubate at 37 C for 24 hours.

SECOND DAY SESSION1. Transfer approximately half of the content of the inoculated broth to an appropriately

labeled

clean tube.

2. Add 0.5 ml (5 drops) Kovac's reagent and mix thoroughly. 3. In te rpre ta t ion :

Cherry-red Layer: Positive Test No Cherry-red Layer: Negative Test

4.

Record the results in the chart.

METHYL RED TEST

PURPOSE

The methyl red (MR) test is performed to learn the following.

Demonstration of the ability of an organism to oxidize glucose with the production 'of highconcentrations of acid end products.

Application of the test in differentiating enteric bacteria.GENERAL CONSIDERATIONS

Glucose, a hexose monosaccharide, is utilized by all enteric bacteria as a major source of nutrition

and energy. Most of these bacteria convert some glucose to acid. However the formation of the end

products depends mainly on the type of enzymes being produced by the bacteria. Certain bacteriaproduce and stabilize low acidic pH (4.0). Other genera enzymatically convert glucose first to

organic acids and them to products such as ethanol and acetylmeth41-carbinol, resulting in aneutral pH. The purpose of MR test, as shown above, is to differentiate between these twogroups of bacteria.

Methyl red is a pH indicator that turns red in the pH range of 4.0. It becomes yellow at pH 6.0 and

above. A red color is indicative of a positive test that means large amount of acids were produced

by bacteria growing in the glucose-containing broth. A negative reaction is shown by a yellow ororange color, indicating less acid production.

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MATERIAL:

CULTURES

24-hour cultures ofE. coil, Ent. aerogenes, and S. typhi.

MEDIA

Clark's broth, one tube each for the given cultures.

REAGENTMethyl red indicator.

EQUIPMENTBunsen burner, 13x100 mm test tubes, and wire loop.

PROCEDURE:

FIRST DAY SESSION

1. Mark the given tubes of Clark's broth with the codes of the cultures and inoculate themrespectively with the help of sterile wire loop.

2. Incubate at 37 C for 24 hours.SECOND DAY SESSION

1. Transfer 1.0 ml of the inoculated Clark's broth in a clean test tube. This tube should belabeled with the name of bacterium used.

2. Add 4 to 5 drops of methyl red reagent and roll the tube between your palms to mix thecontents.

3. Observe the methyl red test culture and interprete the results.

Red coloration : Positive test Yellow coloration : Negative test

4. Record the results in the chart.VOGESPROSKAUER TEST

PURPOSE

By performing Voges-Proskaucr (VP) test, you will learn the following.

Demonstration of the ability of a bacterium to metabolize glucose and produce non-acidic orneutral end products, most important of which is acetylmethylcarbinol.

Application of the test in differentiating enteric bacteria.

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The VP test is always performed along with MR test to differentiate between closely relatedenteric bacteria. The bacteria are grown in Clark's broth, same as used for MR test. Most entericsinitially convert glucose to pyruvic acid which is further metabolized through a number of metabolicpathways, depending upon the types of bacteria. One group of bacteria follows a pathway that leadsto the conversion of pyruvic acid to acetylmethylcarbinol or acetoin. The Klebiella-Enterobacter-Hafnia-Serratia group produce acetylmethylcarbinol (AMC) as the chief end product of glucosemetabolism.

Glucose ______ Pyruvic acid ___ AMC

It has been stated by many workers that AMC is an intermediate stage in the conversion of

pyruvic acid to 2, 3-butanediol. Thus, it may be said that AMC is a precursor to 2, 3-butanediol.

Detection of AMC is carried out by using Barritt's reagent, consisting of a mixture of

alcoholicalpha-naphthol and 40% potassium hydroxide solution. In the presence of atmospheric oxygen and

40% potassium hydroxide AMC is oxidized to diacetyl. This reaction is promoted by alpha-

naphthol which acts as a catalyst. diacetyl reacts with guanidine nucleus of arginine that is present

in the peptone of the Clark's broth. As a result, a pink or red color appears.

AMC + alpha-naphthol 40% KOH Diacetyl + 2H20

02

Diacetyl + guanidine nucleus .Pink/red complex.

MATERIAL:

CULTURES

24-hour cultures ofE. coli , S. typhi andEnterobacter aerogenes.

MEDIA

Clark's broth, one tube each for the given cultures.

REAGENTS

Barritt's reagent A (5% alpha-naphthol) and Barritt's reagent B (40% potassium hydroxide)

EQUIPMENT

Bunsen burner, 13x100 mm test tubes, and wire loop.

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PROCEDURE

FIRST DAY SESSION

Refer to the procedure of MR test.

SECOND DAY SESSION

1. Transfer 1 ml of the inoculated Clark's broth in a clean test tube.2. Add 0.6 ml of Barritt's reagent A followed by 0.2 ml of Barritt's reagent B.

3. Shake the tube gently to expose the contents to atmospheric oxygen.4. Let the tube to remain undisturbed for 10-15 minutes.5. Observe and interprete the results.

Development of a pink or: Positive testred color 15 minutes after addition of the

reagents.

No appearence of pink or red colour : Negative test6. Record the results in the chart.


Like all living organisms, the bacteria also require certain energy and nutritional sources. Carbon

is one of the basic nutrient and also a major energy source for majority of bacteria. In the

absence of glucose or lactose, some bacteria can utilize sodium citrate as the sole carbon source.This ability of bacteria depends upon the synthesis of transport enzyme called citrate permease.It facilitates the transport of sodium citrate from the environment across the cytoplasmic

memberane and into the cell. Within the cell, citrate is acted on by the enzyme citritase, also

known as citrase or citrate demolase.

The medium used for the detection of citrate utilization is a synthetic medium known as SimmonCitrate Agar. It contains sodium citrate as the sole source of carbon and ammonium phosphate as

CITRATE UTILIZATION TEST

PURPOSEBy performing this test, you will learn the following.

Demonstration of the capability of bacteria to utilize citrate as a sole carbon source.

Differentiation amon enteric bacteria.

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the sole source of nitrogen. Another significant ingredient of Simmon Citrate Agar is the pH

indicator bromothymol blue. This indicator is green at neutral pH but turns blue when the pHbecomes 7.6 or above.

The citrate-utilizing bacteria cause alkalinization since utilization of citrate leads to the

generation of alkaline end products including sodium hydroxide, sodium carbonate, andammonium hydroxide. Under such alkaline condition, the bromothymol blue indicator

incorporated into the medium turns from green to deep blue.

Citrate positive bacteria are identified on Simmon Citrate Agar by the development of blue,

coloration accompanied by visible colonial growth. Citrate negative bacteria, however, fail to

grow and change the color of the medium; the color remains green.

MATERIAL:

CULTURES

24-hour cultures ofE. coli, S. typhi, andEnt. aerogenes.

MEDIA

Simmon citrate agar, one slope for each given culture.

EQUIPMENT

Bunsen burner and wire loop.

PROCEDURE:

FIRST DAY SESSION

1. Label each of the Simmon citrate agar slope with the codes of the cultures to beinoculated.

2. Using aseptic technique and sterile wire loop, inoculate the appropriately labeled Simmonslopes with its bacterium by stabbing into the medium, then zigzagging the loop across thesurface of the slope (Stab-and-Streak method).

3. Incubate all the cultures at 37 C for 24 hour.

SECOND DAY SESSION

1. Observe the Simmon citrate agar slope culture. Compare the inoculated slopes with

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uninoculated

control to acertain the outcome of the exercise.

2. Interprete the results.

Blue coloration accompanied:Positive by colonial growth.

No change in the color of:Negative the medium and nogrowth.

4. Record your results in the chart.

OBSERVATIONS:

BACTERIUM INDOLE METHYL RED VOGES-PROSKAUER CITRATE

E. coli

S. typhi

Ent. Aerogenes

RESULTS:

_____________________________________________________________________

_____________________________________________________________________

_____________________________________________________________________

_____________________________________________________________________

POST TEST

COMPLETION

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1. A broth used for methyl red test.___________________________

2. The major intermediate product in the tryptophan degradation.___________________________

3. The reagent used to detect indole.___________________________

4. A test for the detection of acetylmethylcarbinol.___________________________

5.

The other name of AMC.

___________________________

6. The major carbohydrate required for MR test.___________________________

7. The pH indicator incorporated into Simmon citrate agar.___________________________

8. The medium used for indole test.___________________________

9. The ingredient of Kovacs reagent that reacts with indole.___________________________

10.Initial pH of Simmon citrate agar.___________________________

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