Laboratory Exercises in Microbiology - part 10 pps

Laboratory Exercises in Microbiology, Fifth Edition 427 A P P E N D I X B Metric and English Measurement Equivalents The Metric System The metric system comprises three basic units of me

Step #1 1 ml of 10,000 mg/ml stock solution protein  49 ml of diluent  1:50 dilution  200 mg/ml

Step #2 1 ml of 200 mg/ml  9 ml of diluent  1:10 dilution  20 mg/ml

Step #3 1 ml of 20 mg/ml  9 ml of diluent  1:10 dilution  2 mg/ml

To check to make sure the correct dilution was made:

95% 38 ml  _ 38X 950 X  25%

Thus, the dilution factor is 20,000.

Step #1 1 ml of 10,000
g/ml  19 ml of diluent  1:20 dilution  500
g/ml (10,000/20  500)

Step #2 1 ml of 500
g/ml  99 ml of diluent  1:100 dilution  5
g/ml (500/100  5)

Step #3 1 ml of 5
g/ml  9 ml of diluent  1:10 dilution  0.5
g/ml (5/10  0.5)

To check to make sure the correct dilution was made:

1 4

10 40 1:5 dilution0.1 0.4

1 19

10 190 1:20 dilution0.1 1.9

1 7

5 35 1:8 dilution0.5 3.5

1 14

5 70 1:15 dilution0.5 7.0

Problem 14

The first step is to establish the initial dilution as follows:

128

 324

1:32 is the first step dilution, the second is 1:4

1 ml of serum  31 ml of diluent  1:32 (individual dilution)

1 ml of the 1:32 dilution  3 ml of diluent  1:4 (individual dilution)

To check to make sure the dilution was correctly made: 32  4  128

Appendix A Dilutions with Sample Problems 425

Problem 15

We can obtain a 1:3000 dilution in 3 steps by using 1:30 and 1:10 dilutions

3,000

_  1,5002

1 ml of serum  29 ml of diluent  1:30 (individual dilution)

1 ml of 1:30 dilution  9 ml of diluent  1:10 (individual dilution)

1 ml of 1:10  9 ml of diluent  1:10 (individual dilution)

To check to make sure the dilution was correctly made: 30  10  10  3,000

Laboratory Exercises in

Microbiology, Fifth Edition

427

A P P E N D I X B

Metric and English Measurement Equivalents

The Metric System

The metric system comprises three basic units of

measure-ment: distance measured in meters, volume measured in

liters, and mass measured in grams In order to designate

larger and smaller measures, a system of prefixes based on

multiples of ten is used in conjunction with the basic unit of

measurement The most common prefixes are

The English System

The measurements of the English system used in the United

States unfortunately are not systematically related For

ex-ample, there are 12 inches in a foot and 3 feet in a yard

Quick conversion tables for the metric and English systems

are listed below

1 square kilometer (km2) or 1,000,000 m2or 100 ha =

247 A or 0.3861 mi2

The Number of: Multiplied by: Equals:

square centimeters 0.16 square inchessquare meters 1.2 square yardssquare kilometers 0.4 square milesEnglish to Metric

The Number of: Multiplied by: Equals:

square inches 6.5 square centimeterssquare feet 0.09 square meterssquare yards 0.8 square meterssquare miles 2.6 square kilometers

The Number of: Multiplied by: Equals:

English to Metric

1 cubic ft (ft3) or 1,728 in3= 28,317 cm3

or 0.02832 m3

1 cubic yard (yd3) or 27 ft3= 0.7646 m3

The Number of: Multiplied by: Equals:

Units of Liquid Capacity

Metric to English

1 milliliter (ml) or 1 cm3= 0.06125 in3or 0.03 fl oz

1 liter or 1,000 ml = 2.113 pt or 1.06 qt or 0.264 U.S gal

The Number of: Multiplied by: Equals:

428 Appendix B Metric and English Measurement Equivalents

fluid ounce = 1.041 British fluid ounces The British pint, quart, and gallon = 1.2 U.S pints, quarts, and gallons, respectively To convert these U.S fluid measures, multiply by 0.8327.

Laboratory Exercises in

Microbiology, Fifth Edition

Transmission−Absorbance Table for

A logarithm is the exponent of 10, indicating the power to

which 10 must be raised to produce a given number Since

1 is 100and 10 is 101, it is evident that the numbers

be-tween 1 and 10 must be greater than 100 Likewise,

num-bers between 10 and 100 must be greater than 101but less

than 102 These numbers will then have fractional

expo-nents expressed as mixed fractions If they are in fractional

forms, they present difficulties in addition or subtraction, so

it is best to express them as a decimal; for example,

100.3010

A number written in the form b nis said to be in

expo-nential form where b is the base and nis a logarithm For

example, in the following equation,

N = b n

the number N is equal to the base b to the exponent n Let

us say that b is equal to 2 and nis equal to 4 Written in

ex-ponential form, we would have 24 Two to the fourth power

equals 16

In logarithmic form, we would write the log of N to the

base b is n (log b N = n) So if we take 24= 16 and place it

in logarithmic form, we would have

log216 = 4

In the following tables, the logarithms are located in the

body of the table, and the numbers from 1.0 to 9.9 are given

in the left-hand column and the top row For example, to

lo-cate the logarithm of 4.7, read down the left-hand column to

47 and across the column to 0 to find 0.6721 (in the table,

the zero and the decimal point are omitted for convenience)

Finally, the following relationships should be bered when working with logarithms:

remem-log 1 = remem-log 100 = 0log 10 = log 101 = 1log 100 = log 102 = 2log 1,000 = log 103 = 3log 10,000 = log 104 = 4log 0.1 = log 10–1= –1log 0.01 = log 10–2= –2log 0.001 = log 10–3= –3log 0.0001 = log 10–4= –4Logarithms are particularly useful in graphical rela-tions that extend over a wide range of values since theyhave the property of giving equal relative weight to all parts

of the scale This is valuable in “spreading out’’ the valuesthat would otherwise be concentrated at the lower end ofthe scale; for example, in graphing the growth of microbialpopulations in a culture versus time Logarithms are alsoused in pH calculations

430

A P P E N D I X D

Logarithms

pH is a measure of hydrogen ion (H+) activity In dilute

so-lutions, the H+activity is essentially equal to the

concentra-tion In such instances, pH = –log [H+] The pH scale

ranges from 0 ([H+] = 1.00M) to 14 ([H+] = 10–14M)

A pH meter should be used for accurate pH

determina-tions, observing the following precautions:

1 Adjust the temperature of the buffer used for pH

meter standardization to the same temperature as the

sample Buffer pH changes with temperature; for

ex-ample, the pH of standard phosphate buffer is 6.98 at

0°C, 6.88 at 20°C, and 6.85 at 37°C

2 It is important to stir solutions while measuring their

pH If the sample is to be stirred with a magnetic

mixer, stir the calibrating buffer in the same way

3 Be sure that the electrodes used with tris buffers are

recommended for such use by the manufacturer This

is necessary because some pH electrodes do not giveaccurate readings with tris (hydroxymethyl)aminomethane buffers

In instances where precision is not required, such as

in the preparation of routine media, the pH may bechecked by the use of pH indicator solutions By theproper selection, the pH can be estimated within ± 0.2 pHunits Some common pH indicators and their useful pHranges are listed in the following table All of the belowindicators can be made by (1) dissolving 0.04 g of indica-tor in 500 ml of 95% ethanol, (2) adding 500 ml of dis-tilled water, and (3) filtering through Whatman No 1 fil-ter paper Indicators should be stored in a dark, tightlyclosed bottle

All of the above indicators can be made by (1) dissolving 0.04 g of indicator in 500 ml of 95% ethanol, (2) adding 500 ml of distilled water, and (3) filtering through Whatman

No 1 filter paper Indicators should be stored in a dark, tightly closed bottle.

A P P E N D I X F

Scientific Notation

Microbiologists often have to deal with either very large or

very small numbers, such as 5,550,000,000 or 0.00000082

The mere manipulation of these numbers is cumbersome

As a result, it is more convenient to express such numbers in

scientific notation (standard exponential notation)

Sci-entific notation is a set of rules involving a shorthand

method for writing these numbers and performing simple

manipulations with them Scientific notation uses the fact

that every number can be expressed as the product of two

numbers—one of which is a power of the number of ten

Numbers greater than one can be expressed as follows:

In the above notations, the exponent to which the ten is

raised is equal to the number of zeroes following the one.

Numbers less than one can be expressed as follows:

In the above notations, the number of the negative exponent

to which ten is raised is equal to the number of digits to the

right of the decimal point

Numbers that are not an exact power of ten can also be

dealt with in scientific notation For example, a number

such as 1234, which is greater than one, can be expressed

in the following ways:

123.4 × 0.001The same numbers can be expressed in scientific notation

The answer is written as 12.5 × 103 It can also be written

as 1.25 × 104 These same two steps are done in every case

of multiplication, even with numbers less than one For ample, to multiply 0.5 × 0.25:

ex-(5× 10–1)× (2.5 × 10–1)

= 12.5 × 10–2

= 1.25 × 10–1= 0.125When multiplying numbers greater than one by numbersless than one, express the numbers in convenient form,multiply the first part, add the exponents of the second part,and then express the answer in scientific notation For ex-ample, multiply 0.125 × 5,000:

(1.25× 10–1)= (5 × 103)= 6.25 × 102

Laboratory Exercises in

Microbiology, Fifth Edition

Companies, 2002

Appendix F Scientific Notation 433

When adding a negative number to a positive number,

al-ways subtract the negative number from the positive number

Dividing in scientific notation is similar to multiplying

exponent:

103– 102= 101

The answer in scientific notation is expressed as 0.5 × 101.Always remember that when you subtract one negativenumber from another negative number, you add the num-bers and express the answer as a negative number Whensubtracting a negative number from a positive number, it isthe same as adding a positive number to a positive number

To subtract a positive number from a negative number, addthe positive number to the negative number and express theanswer as a negative number

Microbiologists use scientific notation continuously.For example, in this laboratory manual, it is used to de-scribe the number of bacteria in a population and to expressconcentrations of chemicals in solution, of disinfectants,and of antibiotics

A P P E N D I X G *

Identification Charts

*The identification charts presented in this appendix are based on rapid

test systems At times these test results may differ from results obtained

with so-called “conventional” tests.

434

a(Figures indicate the percentage of positive reactions

Escherichieae

Proteeae

Laboratory Exercises in

Microbiology, Fifth Edition

Companies, 2002

Appendix G Identification Charts 437

Chart II Characterization of Enterobacteriaceae—The Enterotube II System

E S enteritidis bioserotype Paratyphi A and some rare biotypes may be H2 S negative.

F S typhi, S enteritidis bioserotype Paratyphi A and some rare biotypes are citrate-negative and S cholerae-suis is usually delayed positive.

G The amount of gas produced by Serratia, Proteus, and Providencia alcalifaciens is slight; therefore, gas production may not be evident in the ENTEROTUBE II.

H S enteritidis bioserotype Paratyphi A is negative for lysine decarboxylase.

I S typhi and S gallinarium are ornithine decarboxylase-negative.

J The Alkalescens-Dispar (A–D) group is included as a biotype of E coli Members of the A–D group are generally anaerogenic, non-motile and do not ferment lactose.

K An occasional strain may produce hydrogen sulfide.

L An occasional strain may appear to utilize citrate.

Copyright © Becton Dickinson Microbiological Systems Reprinted by permission.

Reactions Glucose

De am in ase Urea

Citrate Groups

438 Appendix G Identification Charts

Chart III Characterization of Oxidative-Fermentative Gram-Negative Rods

H

2S

OF Anaerobic Dextrose

Laboratory Exercises in

Microbiology, Fifth Edition

439

Reagents and stains appear in this appendix as the authors

have presented the material in the individual laboratory

ex-ercises and are listed in alphabetical order When

neces-sary, methodology is given with the reagents, stains, or

tests The detailed procedures, however, are presented in

the exercise in which their use is discussed

Acid-Alcohol (for Ziehl-Neelsen stain)

Concentrated hydrochloric acid 3 ml

Solution B: 40 g of potassium hydroxide in

100 ml of water Store in the refrigerator

Bile Solubility Test (10% bile)

Sodium deoxycholate 1 g

Sterile distilled water 9 ml

To test for bile solubility, prepare two tubes, each

con-taining a sample of fresh culture (a light suspension of

the bacterium in buffered broth, pH 7.4) To one tube

add a few drops of a 10% solution of sodium

deoxy-cholate The same volume of sterile physiological

saline is added to the second tube If the bacterial cells

are bile soluble, the tube containing the bile salt will

lose its turbidity in 5 to 15 minutes and show an

in-crease in viscosity

Cleaning Solution for Glassware Strong:

Potassium dichromate 20 gDistilled water 200 mlDissolve dichromate in water; when

cool, add very slowly:

Concentrated sulfuric acid 9 parts2% aqueous potassium dichromate 1 part

Copper Sulfate Solution (20%)

Copper sulfate (CuSO4 5H2O) 20 gDistilled water 80 ml

Crystal Violet Capsule Stain (1%)

Crystal violet (85% dye content) 1 gDistilled water 100 ml

Decolorizers (for Gram stain)

1 Intermediate agent, 95% ethyl alcohol

2 Fastest agent, acetone

3 Slowest agent, acetone-isopropyl alcohol(isopropyl alcohol, 300 ml; acetone, 100 ml).For the experienced microbiologist, any one

of the three decolorizing agents will yieldgood results

Diphenylamine Reagent (for the nitrate test)

Working in a fume hood, dissolve 0.7 g ofdiphenylamine in a mixture of 60 ml ofconcentrated sulfuric acid and 28.8 ml of distilledwater Allow to cool Slowly add 11.3 ml ofconcentrated hydrochloric acid After the solutionhas stood for 12 to 24 hours, some of the base willseparate This indicates that the reagent is saturated

A P P E N D I X H

Reagents, Solutions, Stains, and Tests

440 Appendix H Reagents, Solutions, Stains, and Tests

Gram’s Iodine (Lugol’s)

According to the ASM Manual for Clinical

Microbiology, dissolve 2 g of potassium

iodide in 300 ml of distilled water and

then add 1 g of iodine crystals Rinse the

solution into an amber bottle with the

remainder of the distilled water Discard

when the color begins to fade

Add solution A to solution B Let stand

for a day, then filter If the crystal violet

is too concentrated, solution A may be

diluted as much as 10 times

(B) Gram’s Iodine Solution (mordant)

For a working solution, dilute stock solution 1/10

(10ml of stock safranin to 90 ml of distilled

water)

India Ink (for capsule stain)

Mix the specimen with a small drop of India ink

on a clean slide If the India ink is too dark, dilute

it to 50% with distilled water

Kinyoun Acid-Fast Stain

(A) Kinyoun Carbolfuchsin

Kovacs’ Reagent (for the indole test)

N-amyl or isoamyl alcohol 150 ml

Concentrated hydrochloric acid 50 ml

p-dimethylaminobenzaldehyde 10 g

Working in a fume hood, dissolve the aldehyde inalcohol and then slowly add the acid The dryaldehyde should be light in color Alcohols thatresult in indole reagents that become deep brownshould not be used Store in a dark bottle with aglass stopper in a refrigerator when not in use

Malachite Green Solution (for endospore stain)

Malachite green oxalate 5 gDistilled water 100 ml

Methylene Blue (Löffler’s alkaline) Solution A: Dissolve 0.3 g of methylene blue

(90% dye content) in 30 ml of 95% ethyl alcohol

Solution B: Dissolve 0.01 g of potassium

hydroxide in100 ml of distilled water

Mix solutions A and B Filter with Whatman

No 1 filter paper before use

Methylene Blue Stain (simple staining)

Methylene blue 0.3 gDistilled water 100.0 ml

Methyl Red Reagent (for detection of acid)

Methyl red 0.1 g95% ethyl alcohol 300 mlDissolve the dye in alcohol and add sufficientdistilled water to make 500 ml Positive tests arered-orange, and negative tests are yellow

Naphthol, Alpha (for the Enterotube II System)

5%α-naphthol in 95% ethyl alcohol

Nessler’s Reagent (for the ammonia test)

Working in a fume hood, dissolve 50 g of potassiumiodide in 35 ml of cold (ammonia-free) distilled water.Add mercuric chloride drop by drop until a slightprecipitate forms Add 400 ml of a 50% solution ofpotassium hydroxide Dilute to 1 liter, allow to settle,and decant the supernatant for use Store in a tightlyclosed dark bottle

Alternate procedure:

Solution A:

Mercuric chloride 1 gDistilled water 6 mlDissolve completely

Solution B:

Potassium iodide 2.5 gDistilled water 6 ml

Solution C:

Potassium hydroxide 6 gDistilled water 6 mlDissolve solution C completely and add to themixture of solutions A and B Add 13 ml ofdistilled water Mix well and filter throughWhatman No 1 filter paper before use Store in adark, stoppered bottle

Laboratory Exercises in

Microbiology, Fifth Edition

Appendix H Reagents, Solutions, Stains, and Tests 441

Nigrosin Solution (Dorner’s, for negative staining)

Water-soluble nigrosin 10.0 g

Distilled water 100.0 ml

Formalin (40% formaldehyde) 0.5 ml

Gently boil the nigrosin and water approximately

30 minutes Add 0.5 ml of 40% formaldehyde as a

preservative Filter twice through Whatman No 1

filter paper and store in a dark bottle in the

refrigerator

Nitrate Test Reagent (see under diphenylamine)

Nitrite Test Reagents (Caution—solution B may be

carcinogenic Use safety precautions such as the

avoidance of aerosols, mouth pipetting, and contact

with skin.)

(A) Solution A: Dissolve 8 g of sulfanilic acid in

1 liter of 5 N acetic acid (1 part glacial acetic acid

to 2.5 parts distilled water)

(B) Solution B: Dissolve 6 ml of N,

N,-dimethyl-1-naphthylamine in 1 L of 5 N acetic acid

DO NOT MIX SOLUTIONS

Oxidase Test Reagent

Mix 1 g of dimethyl-p-phenylenediamine

hydrochloride in 100 ml of distilled water This reagent

should be made fresh daily and stored in a dark bottle

in the refrigerator

O-nitrophenyl-β-D-Galactoside (ONPG)

0.1 M sodium phosphate buffer 50.0 ml

ONPG (8 ×10–4M) 12.5 mg

Phosphate Buffers

Stock buffers:

Alkaline buffer, 0.067 M Na2HPO4solution

Dissolve 9.5 g of Na2HPO4in 1 liter of distilled

water

Acid buffer, 0.067 M NaH2PO4solution Dissolve

9.2 g of NaH2PO4 H2O in 1 liter of distilled

water

Buffered water (pH 7.0 to 7.2)

Acid buffer (NaH2HPO4) 39 ml

Alkaline buffer (Na2HPO4) 61 ml

Distilled water 900 ml

BE SURE GLASSWARE IS CLEAN Buffered water,

if sealed, is stable for several weeks

Physiological Saline

Dissolve 8.5 g of sodium chloride in 1 liter of

distilled water (0.85%) or 9 g in 1 liter of distilled

water (0.9%)

Physiological Saline (Buffered)

Sodium chloride (0.85%; 8.5 g in 1 liter of

distilled water) is buffered to pH 7.2 with 0.067 M

potassium phosphate mixture

Add water to 1 liter

Triton X-100 Stock Solution (10%)

Triton X-100 10 mlDistilled water 90 mlMix and store in a tightly stoppered bottle at roomtemperature; the solution will keep indefinitely

Trommsdorf’s Reagent (for the nitrite test)

Working in a fume hood with a beaker on a hotplate, slowly add, with constant stirring, 100 ml of

a 20% aqueous zinc chloride solution to a mixture

of 4 g of starch in water Continue heating untilthe starch is completely dissolved and the solution

is clear Dilute with water and add 2 g ofpotassium iodide Dilute to 1 liter with distilledwater, filter once through Whatman No 1 filterpaper, and store in a capped, dark bottle

442 Appendix H Reagents, Solutions, Stains, and Tests

Solution B:

Stain: 7.5 g of silver nitrate (AgNO3) in 150 ml of

distilled water While working in a fume hood,

add concentrated NH4OH dropwise to 140 ml of

the silver nitrate solution while it is being stirred

on a magnetic mixer A brown precipitate will

form at the start of NH4OH addition Enough

NH4OH should be added so that the brown

precipitate just dissolves Finally, add 5% silver

nitrate dropwise until a faint cloudiness persists

This solution should be stored at 5°C in an

aluminum foil-covered bottle until used

Ziehl-Neelsen Acid-Fast Stain

(A) Solution A: Dissolve 0.3 g of basic fuchsin

(90% dye content) in 10 ml of 95% ethyl alcohol

Solution B: Dissolve 5 g of phenol in 95 ml of

distilled water

Mix solutions A and B Note: Add either 1 drop ofTergitol No 4 per 30 ml of carbolfuchsin or 2drops of Triton X-100 per 100 ml of stain for use

in the heatless method Tergitol No 4 and Triton

X act as detergents, emulsifiers, and wettingagents

(B) Acid-alcohol, 3%

Concentrated hydrochloric acid 3 ml95% alcohol 97 ml

Laboratory Exercises in

Microbiology, Fifth Edition

Companies, 2002

Sterilization of all tubed media is accomplished at 15 lb

pressure (121°C) for 15 minutes unless otherwise specified

Longer sterilization times will be required for large volumes

of media Most of the media are available commercially in

powdered form, with specific instructions for their

prepara-tion and sterilizaprepara-tion

Sources of Microbiological Media

In addition to making media from commercially prepared

supplies, companies such as Oxoid Unipath, 800 Proctor

Av-enue, Ogdensburg, NewYork 13669-2205; Scott

Laborato-ries, West Warwick, Rhode Island 02893 and Carson,

Califor-nia, 90746; Fisher Scientific, 711 Forbes Avenue, Pittsburgh,

Pennsylvania 15219; The Scientific Products Division of

Bax-ter Healthcare Corporation, 1430 Waukegan Road, McGrawPark, Illinois 60085; Wards Natural Science Establishment,

5100 West Henrietta Road, P.O Box 92912, Rochester, NewYork; and Carolina Biological Supply, 2700 York Road,Burlington, North Carolina 27215 can supply most of themedia used in this manual already prepared in tubes, bottles,and plates Some offer special services for diagnostic media

Actidione (Cycloheximide) Agar (pH 5.5)

Glucose 50.0 gAgar 15.0 gPancreatic digest of casein 5.0 gYeast extract 4.0 gPotassium dihydrogen phosphate 0.5 gPotassium chloride 0.42 gCalcium chloride 0.12 gMagnesium sulfate 0.12 gBromcresol green 22.0 mgActidione (cycloheximide) 10.0 mgFerric chloride 2.5 mgDistilled water 1,000.0 ml

Agar, Noble

Noble agar is carefully washed agar that is purified and sentially free from impurities It is used in electrophoreticprocedures, nutritional studies, and wherever an agar of in-creased purity is needed

es-Ammonium Sulfate API Broth (pH 7.5)

Bacto yeast extract 1 gAscorbic acid 0.1 gSodium lactate 5.2 gMagnesium sulfate 0.2 gDipotassium phosphate 0.01 gFerrous ammonium sulfate 0.1 gSodium chloride 10.0 gDistilled water 1,000.0 ml

Azotobacter Nitrogen-Free Broth (pH 7.2)

Dipotassium phosphate 1.0 gMagnesium sulfate 0.2 gSodium chloride 0.2 gFerrous sulfate 5.0 mgDistilled water 1,000.0 ml

443

A P P E N D I X I

Culture Media

444 Appendix I Culture Media

Bile Esculin Agar (pH 6.8)

Note: Dissolve the above ingredients and autoclave Cool

the sterile blood agar base to 45° to 50°C and aseptically

add 50 ml of sterile, defibrinated blood Mix thoroughly

and then dispense into plates while a liquid Blood agar

base for use in making blood agar also can be purchased

A combination of hemoglobin and a commercial nutrient

supplement can be used in place of defibrinated blood

Bottom Agar (pH 7.0)

Use 12-ml sterile nutrient agar pours to prepare plates

Brain-Heart Infusion Agar (pH 7.4)

Calf brains, infusion from 200.0 g

Beef hearts, infusion from 250.0 g

15 minutes or until a chocolate color develops

Cystine Tryptic Agar (pH 7.3)

Tryptose 20.0 gL-cystine 0.5 gSodium chloride 5.0 gSodium sulfite 0.5 gAgar 2.5 gPhenol red 0.017 gDistilled water 1,000.0 mlAfter autoclaving and cooling to 50°C, add appropriateBacto Differentiation Disk Carbohydrate (e.g.,dextrose, fructose, maltose, sucrose) Allow to coolunslanted in an upright position

Deoxyribonuclease (DNase Test) Agar (pH 7.3)

Deoxyribonucleic acid 2.0 gPhytone peptone 5.0 gSodium chloride 5.0 gTrypticase 15.0 gAgar 15.0 gDistilled water 1,000.0 ml

Endo Agar (pH 7.5)

Peptone 10.0 gLactose 10.0 gDipotassium phosphate 3.5 gSodium sulfite 2.5 gBasic fuchsin 0.4 gAgar 15.0 gDistilled water 1,000.0 ml

Enriched Nitrate Broth

See Nitrate Broth

Eosin-Methylene Blue (EMB) Agar (pH 7.2)

Peptone 10.0 gLactose 5.0 gSucrose 5.0 gDipotassium phosphate 2.0 gAgar 13.5 gEosin Y 0.4 gMethylene blue 0.06 gDistilled water 1,000.0 ml

Eugon Agar (pH 7.0)

Tryptose 15.0 gSoytone 5.0 gDextrose 5.0 gL-cystine 0.2 gSodium chloride 4.0 gSodium sulfite 0.2 gAgar 15.0 gDistilled water 1,000.0 ml

(A 1/1,000 dilution of merthiolate can be added as a

preservative Dispense in appropriate dishes.)

Prepare solutions of individual amino acids to give

2 mg/ml and filter sterilize

Lactose Fermentation Broth (1 ⴛ and 2ⴛ, pH 6.9)

Beef extract 3.0 gPeptone 5.0 gLactose 5.0 gDistilled water 1,000.0 mlNote: For the 2×, use twice the ingredients

Lauryl Tryptose Broth (pH 6.8)

Tryptose 20.0 gLactose 5.0 gPotassium phosphate, dibasic 2.75 gPotassium phosphate, monobasic 2.75 gSodium chloride 5.0 gSodium lauryl sulfate 0.1 gDistilled water 1,000.0 ml

Levine EMB Agar (pH 7.1)

Peptone 10.0 gLactose 10.0 gDipotassium phosphate 2.0 gAgar 15.0 gEosin Y 0.4 gMethylene blue 0.065 gDistilled water 1,000.0 ml

Litmus Milk

Skim milk powder 100.0 gLitmus 0.75 gDistilled water 1,000.0 mlNote: Autoclave at 12 lb pressure for 15 minutes

Löwenstein–Jensen Medium

Asparagine 3.6 gMonopotassium phosphate 2.4 gMagnesium sulfate 0.24 gMagnesium citrate 0.6 gPotato flour 30.0 gMalachite green 0.4 gDistilled water 600.0 ml

Lysine Iron Agar (pH 6.7)

Peptone 5.0 gYeast extract 3.0 gDextrose 1.0 g