TAMILNADU VETERINARY AND ANIMAL SCIENCES UNIVERSITY

Training Manual On
BACTERIOLOGICAL TECHNIQUES USED IN ANIMAL DISEASE DIAGNOSIS
(Training to the Laborator te!hni!ian" #ro$ Ani$a% H&"ban'r 'e(art$ent) Go*ern$ent o# Bh&tan+

Dr, -ari$a% Ro Dr, S,S&re"h.annan Dr, G, Ba%a.ri"hnan

CENTRAL UNIVERSITY LABORATORY

CENTRE /OR ANIMAL HEALTH STUDIES MADHAVARAM MIL0 COLONY) CHENNAI 1 233 345, Te%e(hone6 377184445495: /a;6 3771844454<<: E$ai%6 !&%!ah"=re'i##$ai%,!o$

INDEX 1

S.No 1.

#.

4.

&. (.

+. /. 1.

Title Sterilization 1.1 Physical Method 1. !he"ical "ethod of Sterilization 1.# $aseo%s "ethod 1.4 'ilters !%lt%re and identification of )acteria .1 *acteriological "edia and different "edia to )e %sed . Sa",le ,rocessing and c%lt%re flo- chart .# *acterial gen%s identification flo- chart .4 Strea.ing "ethods .& *acterial colony "or,hology .( Maintenance and ,reser0ation of ,%re c%lt%re Staining of )acteria #.1 Pre,aration of !lean slide #. $ra"s Staining #.# 2lternati0e test for $ra"s Staining #.4 3iehl - Neelsen 43N5 Staining #.& Staining Techni6%e for 2nthra7 )acilli Secondary le0el identification of *acteria )y )ioche"ical tests 4.1 *ioche"ical tests ,roced%res and o)ser0ations 4. $ra" negati0e )acteria *ioche"ical tests 4.# $ra" Positi0e )acteria *ioche"ical tests *ioche"ical tests 2nti"icro)ial S%sce,ti)ility testing (.1 $eneral g%idelines (. Inter,retation (.# 2nti"icro)ial sensiti0ity chart !olifor" co%nt in -ater sa",les 8a)oratory test for f%ng%s Diseases and Materials to )e collected fro" 8i0estoc. and ,o%ltry 9eferences

Page No. 1-4 4 4-& &-( + -1 1# 14 1&-1( 1+-1/ 11- 1 1 - # # #- 4 4 (-## #4 #&-#( #+ #/-#1 #1-41 41-4 4# 44-4( 4+-& &#

01. Sterilization
The explicit identification of etiological agent depends highly on the use of sterile laboratory accessories and procedures. To avoid contaminants, one must remove or kill all microorganisms from equipment and media used for microbiological work to eliminate or reduce the possibility of unwarranted contaminants. All the equipments such as glassware, scalpels, needles, forceps, etc., and the media for culturing microorganisms must be sterilized thoroughly using principles of aseptic technique. Sterilization !eans complete destruction of all"forms of microbial life. A sterile ob#ect, in the microbiological sense, is free of all living microorganisms. There is no such thing as a $practically sterile$ or $nearly sterile$% it is either sterile or it is not sterile. Methods of Sterilization &

There are many methods of sterilization but most of them fall under the broad classification of physical, chemical and gaseous ones. 1.1 Physical Methods of Sterilization 2. Dry :eat 'a( 'la"ing ; )mall ob#ects, e.g., inoculating loops and needles that are not easily damaged by heat, can be sterilized by thrusting them into the flame dull"red heating to a temperature high enough to destroy any organisms present upon the surface. 'b( :ot-air sterilization ; *n this method the glass wares are first dried properly, wrapped in brown paper and then exposed to hot"air in electric or gas oven to a temperature of 1+,-. for twohours. This treatment is sufficient for complete sterilization because at this temperature destruction of all living cells and viable spores take place due to destructive oxidation of the cell contents. /niform heating depends upon proper loading in the oven. A further rise in temperature may char the paper or cotton plug. :ot-air o0en is operated by electric power and used for sterilizing glasswares, e.g., 0etri dishes, flasks, tubes, pipettes and other glass wares in microbiological laboratories. The walls of the sterilizer are made of stainless steel or aluminium and are so devised that the heat conduction from inside to outside is completely prevented. The oven consists of a big chamber into which the materials to be sterilized are kept. A fan is set at the bottom of the oven which forces steam of hot dry air circulating through the chamber resulting in rise in chamber"temperature to sterilize the materials. A thermometer is fitted for recording the temperature of the oven. Temperature at 1+,-. sterilizes the glassware1s in a period of two hours.

:<T 2I9 <=EN 1.2xhaust &. 3iffusion 4all 5. 6lass 4ool *nsulation 5

7. Air 8low )ampler 9. Turbo :lower +. !otor 2 sched%le of ti"e and te",erat%re for sterilization -ith dry air is as follo-s;

*. Moist :eat !oist heating is more efficient in sterilization than the dry heating because heat conduction is less rapid, the process takes much longer time and the death rate is lower in dry heat as compared to the moist heat. .ulture media, aqueous solutions, cloths, rubber and other materials that would be destroyed by dry heating are sterilized by moist heating. 8ollowing are the commonly used methods of moist heat sterilization.
Te",erat%re 4?!5 1&, 17, 19, 1+, 1>, 1=, Ti"e 4"in%tes5 7=, 1=, 19, 1&, +, &,

Tyndallization Inter"ittent sterilization

or

.ertain media containing gelatin, milk and sugars get adversely affected by heating

at high temperature and it would, therefore, be prudent to use intermittent 'also called ;fractional;( sterilization with the help of earlier described Arnold )team )terilizer. This method involves heating the material at 1,,-. for 5, minutes on three successive days with incubation periods in between. <esistant spores germinate during the incubation periods% the newly formed vegetative cells get destroyed on subsequent exposure to heat. The disadvantage of this process is that it is time consuming and non"nutrient solution can not be sterilized by this method because resistant spores may not germinate but remain dormant in such solution. 'c( Stea" >nder Press%re ; This method is useful for sterilization of media as well as apparatus. The laboratory apparatus designed to use steam under regulated pressure is called an a%tocla0e. The autoclave is an essential unit of equipment in every microbiological laboratory. Princi,le The steam is allowed to form in the inner cylinder of an autoclave by heating water. The steam pressure inside the cylinder increases with the time of heating and the steam valve 7

is shut off. The pressure developing inside can be read out by a pressure gauge. 6enerally an autoclave is operated at a pressure of approximately 19 lbs?in& '1&1-.(. The time of operation to achieve sterility depends on the nature of the material being sterilized, the type of container, and the volume. 8or example, test tubes of liquid media can be sterilized in 1,"19 minutes at 19 lbs?in& '1&1-.( the same media in 1, litre quantities would require an hour or more at the same temperature for complete sterilization. *n general sterilization is done at variable heat penetration time, constant sterilization and arbitory safety margin time% when volume of the liquid is less than 9, ml taken in thin glass containers, heat penetration time is > minutes, sterilization time is 9 minutes and safety margin is 5 minutes.After the desired time of exposure to steam under pressure, the supply of heat is cut off and steam pressure in the autoclave allowed to come down to zero before opening the lid for removal of sterilized materials.

:orizontal 2%tocla0e

')ource " http ??www.microbiologyprocedure.com(

Sterilization of test t%)es containing li6%id "edia

2xposure periods required for aqueous solution or liquids in various containers affording a reasonable factor of safety for sterilization by autoclaving
.ontainer Test tubes 2rlenmeyer flask )ize 1= x 19, mm 5& x &,, mm 5= x &,, mm 9, ml 9,, ml 1,,, ml &,,, ml 9,, ml 1,,, ml &,,, ml !inutes exposure at &9,"&97@8 '1&1"1&5@.( 15"1> 1&"17 19"&, 1&"17 1>"&& &,"&9 5,"59 &7"&= &9"5, 7,"79

8enwal flask

!ilk"dilution bottle )erum bottle

&,, ml A,,, ml

15"1> 9,"99

1. !he"ical Methods of Sterilization A variety of non"volatile chemicals are generally used in the laboratory to sterilize discarded glassware, desk, hand gloves, etc. The primary ob#ective of the use of such chemicals is to kill potentially dangerous microorganisms present on such articles, and also to reduce the laboratory atmosphere from fungal spores. There are wide varieties of disinfectants, some important examples are as follows .hemical group
:alogens

Bame of .hemical

the
4ork as general disinfectant and sanitizer Antiseptic )kin antiseptic /seful disinfectant for surface sterilization of bench tops, inanimate ob#ects% orgnanomercurials as antiseptics for skin 6ermicidal agents, effective against wide range of microorganisms )kin antiseptic, disinfectant for utensils.

2lcohols :ea0y "etals Phenolic co",o%nds @%arternary a""oni%" co",o%nds Nitrates 2ldehydes

.hlorine .hlorine and its compounds *odine and *odophores 2thyl and *sopropyl !ercuric chloride and organomercurials Cysol, .resol etc.

Alkyl"dimethyl"benzyl" ammonium chloride etc. )ilver nitrate 3isinfectant for the surface of test materials 8ormaldehyde /se as microbiostatic% it is less commonly used because of its irritating vapour

1.# $aseo%s Methods of Sterilization Ethylene o7ide :y far, the most effective and useful gaseous disinfecting agent known is ethylene oxide '.&D7E(. 2thylene oxide is used only in special cases when sterilization is not possible by any other easy method because the gas is explosive and toxic to man. The vapour of ethylene oxide is used under pressure in a special equipment. This vapour is highly toxic to viruses, bacteria and fungal cells and also to the heat resistant spores and endospores. 2thylene oxide is now widely used in hospitals and in industries for sterilizing heat sensitive materials. *n hospitals, it is used for disinfecting chemical respirators, heart"lung machines, opthalmoscopes, etc. Poly"er of for"aldehyde *n recent years, a polymer of formaldehyde, namely, paraformaldehyde is being used as bactericidal, sporicidal and virucidal agent. *ts action is rapid at temperature of 97"9A-. and at relative humidity of =&"A,F.. +

1.4 'ilters; There are different types of filters used in microbiology viz. 1.)eitz filter &. )intered glass filter 5. !embrane filter 'i( Seitz 'ilter 'Asbestos filter(. This filter consists of &"+ mm compressed asbestos fibre filter sheet. A variety of filter sheets containing different pore sizes are available in discs or squares ready for use and work satisfactorily only for a few hours. The medium to be filtered 'sterilized( is poured into the funnel"like structure and drawn through filter sheet by vacuum. 4hen the filtration is complete the filter sheet is discarded and the filtrate is obtained. A modified )eitz filter in which vacuum"drawn filtrate technique has been replaced by centrifugal technique is also used now"a"days where the filter is mounted on a centrifuge which forces the filtrate into the tube. Seits filter 4as)estos filter5 Me")rane 'ilter

')ource "

http ??www.microbiologyprocedure.com(

Diagra""atic graded filtration

re,resentation of ,rinci,les

>

')ource " http ??www.microbiologyprocedure.com( 'ii( Me")rane or Molec%lar 'ilter A new type of filter called Gmembrane$ or ;molecular; filter has been developed in recent years. /nlike bacteriological filters which retain only bacteria, membrane filter retains all forms of microorganisms according to their pore size. 2xamples are syringe filters of porosity && Hm or 79 Hm. )imilar filters are used for media filtration. These filters are made up of biologically inert cellulose esters, and are prepared as circular membranes consisting of millions of pores of uniform and specifically predetermined size. !embrane filters were originally manufactured by the !illipore 8ilter .orporation '/)A( and therefore they are also known as $!illipore; or ;/ltra filters;. . !>8T>9E 2ND IDENTI'I2TI<N <' *2!TE9I2 The role of suitable quality culture media for cultivation of microorganisms cannot be over emphasized. The success in isolation of aetiological agents depends on selective inoculation in specific media. Enly in exceptional cases, an organism could be identified on the basis of its morphological characteristics alone. :ut in most cases other biochemical tests and diagnostic aids are necessary to be performed to identify an etiological agent. .1 *2!TE9I<8<$I!28 MEDI2 Ty,es of "edia :acteriological media can be broadly sub"divided into four categories. 1. $eneralised c%lt%re "edia 6eneralised culture media are routinely employed in a laboratory for primary isolation of an organism. This media will support the growth of many type of bacterium in general. e.g. nutrient broth, nutrient agar, :rain Deart *nfusion ':D*( broth, Tryptose 0hosphate broth etc.. . Enriched "edia =

.ertain organisms do not grow on ordinary nutrient media. They require growth" promoting ingredients such as blood, glucose, serum, egg, etc. The media containing ingredients which enhance their growth"promoting qualities are enriched media e.g. blood agar, chocolate agar and loeffler medium. #. Enrich"ent "edia 2nrichment media are liquid media containing chemical constituents which inhibit some normal flora and allow pathogens which may be present in very small number in the specimen, to grow unhampered and thus enriching them. *solated colonies of these organisms may be obtained by subculturing onto solid media. e.g. )elenite broth, tetra thionate broth for the enrichment of )almonella and listeria enrichment broth for Listeria spp. 4. Selecti0e and differential; )elective and differential media are used to isolate or identify particular organisms. )elective media allow certain types of organisms to grow, and inhibit the growth of other organisms. The selectivity is accomplished in several ways. 8or example, organisms that can utilize a given sugar are easily screened by making that sugar the only carbon source in the medium. En the other hand, selective inhibition of some types of microorganisms can be achieved by adding dyes, antibiotics, salts or specific inhibitors which affect the metabolism or enzyme systems of the organisms. 8or example, media containing potassium tellurite, sodium azide or thallium acetate 'at concentrations of ,.1 " ,.9 g?l( will inhibit the growth of gram"negative bacteria. !edia supplemented with penicillin '9"9, units?ml( or crystal violet '& mg?l( will inhibit the growth of gram"positive bacteria. Therefore, tellurite agar is used for selective isolation of gram"positive organisms, and nutrient agar supplemented with penicillin can be used to select gram negative organisms.

3ifferential media are used to differentiate closely related organisms or groups of organisms. Ewing to the presence of certain dyes or chemicals in the media, the organisms will produce characteristic changes or growth patterns that are used for identification or differentiation. A variety of selective and differential media are used in medical, diagnostic and water pollution laboratories, and in food and dairy laboratories. Dere are few commonly used selective and differential media described below 1. :lood agar '2nriched media( I *t enhance growth and also differentiates haemolytic with non haemolytic organism.

&. !ac .onkey agarI'3ifferential media( " 3ifferentiates lactose fermenting and non lactose fermenting bacteria(. 5. Jylose Cysine 3eoxycholate agar 'JC3( I '3ifferential media( I 3ifferentiates various enteric organisms. 7. )abourauds 3extrose agar ')3A( I )elective isolation of fungus and yeasts 9. !anitol salt agar '!)A( I )elective isolation of )taphylococcus spp. +. 2dwards medium " )elective isolation of )treptococcus :ut in diagnostic bacteriology it is better we could streak the specimen in one generalized medium along with blood agar and two differential ,medium of suspected pathogen e.g. for enteric pathogen we should streak on !ac .onkey and JC3 agar. *lood 2gar; A non"selective medium enriched medium for the isolation and cultivation of many pathogenic and non"pathogenic microorganisms like )taphylococcus, )treptococci etc. The medium is often used to observe different forms of haemolysis from pathogenic microorganisms. !o",osition; *ngredients 6rams?Citre !eat extract 0eptone )odium chloride Agar " " " " 1,., 1,., 9., 19.,

8inal pD +.=K?",.& at 5>-. )uspend 7, g of dehydrated medium in 1 litre of distilled water and boil it to dissolve the suspended agar completely. )terilize by autoclaving at 1&1-. for 19 minutes. 8or the addition of defibrinated blood, cool the autoclaved medium to 79"9,-. and add aseptically +F '9"1,F( of sterile defibrinated blood. .heck for sterility by keeping the plates overnight in incubator. )tore the plates below =-., protected from direct light. )tore dehydrated powder, in a dry place, in tightly"sealed containers at &"&9-.. Princi,le and Inter,retation; !eat extract and 0eptone provide nitrogenous compounds, vitamins, carbon, sulphur and amino acids in :lood Agar :ase. The medium contains sodium chloride for the osmotic balance. :lood Agar :ases are relatively free of reducing sugars, which have been reported to adversely influence the hemolytic reactions of beta"hemolytic streptococci. )heep blood gives best results for 6roup A )treptococci. 4hen horse blood is used, Haemophilus 1,

haemolyticus colonies produce haemolysis and mimic Streptococcus. Daemolytic patterns may vary with the source of animal blood or type of base medium used. )light acidic pD '+.= L ,.&( favours distinct hemolytic reaction and is advantageous for cultivation of )treptococci and 0neumococci. The low pD helps in stabilization of red blood corpuscles and favours the formation of clear haemolysis zone. !hocolate agar ; 0reparation of chocolate agar is same as in blood agar, :ut after the addition of blood, the media is kept in water bath for about 19 minutes 'until haemolysis occurs( at >&. . .hocolate agar containing 1,F sterile defibrinated blood is suited for the isolation of Haemophilus and Neisseria species. Bormally J factor 'Daemin( and M factor 'Bicotinamine Adenine 3inucleotide( is required for the growth of Haemophilus organisms. 4hile the media is kept in water bath at >&., <:.s breaking down takes place and release the required factors in to the medium. 8or the selective isolation of tubercle bacilli, addition of 1 F glycerol and &9 F human blood is recommended. Mac!on.eyAs 2gar !ac.onkey1s agar is a differential plating medium used in the detection and isolation of all types of enteric bacterium. *t is generally used for differentiating strains of Salmonella from the members of the coliform group which ferment lactose sugar% however, the medium supports the growth of all Salmonella and Shigella strains and gives good differentiation between these enteric pathogens and the coliform group. 4hen grown on !ac.onkey1s medium, colonies of coliform bacteria are pink in color and are surrounded by a zone of precipitated bile% Klebsiella organism produce partial pink coloured mucoid ad#oined coalescent colonies% )almonella, )higella and 0roteus organisms produce colour less colonies. These reactions are due to the acid produced by the fermentation of lactose. The acid end"products act on bile salts, and neutral red is absorbed by the precipitated salts. .olonies of these organisms are noncolored and transparent. The growth of 6rampositive organisms is inhibited because of the crystal violet and bile salts in the medium. Eosin Methylene *l%e 2gar 4EM*5 2osin methylene blue agar is a differential medium used for the detection and isolation of 6ram"negative intestinal pathogens. A combination of eosin and methylene blue is used as an indicator and allows differentiation between organisms that ferment lactose and those that do not. )accharose is also included in the medium because certain members of the 2nterobacteria or coliform group ferment saccharose more readily than they ferment lactose. *n addition, methylene blue acts as an inhibitor to 6ram"positive organisms. .olonies of E. 11

coli normally have a dark center and a greenish metallic sheen, whereas the pinkish colonies of Enterobacter aerogenes are usually mucoid and much larger than colonies of E. coli. Mannitol Salt 2gar 4MS25 !annitol salt agar is a selective medium used for the isolation of pathogenic staphylococci. The medium contains mannitol, a phenol red indicator, and >.9F sodium chloride. The high salt concentration inhibits the growth of most bacteria other than staphylococci. En !)A, pathogenic Staphylococcus aureus produces small colonies surrounded by yellow zones. !annitol fermentation is the reason why S. aureus produce yellow coloured colonies due to acid production, which changes the indicator from red to yellow.

Microorganis"s of =eterinary i",ortance and so"e s,ecific "edia %sed

S.No. 1. &. Na"e of the organis"s and disease E.coli .olibacillosis Salmonella spp. ')almonellosis( Media %sed -ith the Media !atalog%e Staining No. of :i"edia and Difco !ac.onkey agar '!,=&A"9,6(, 2!: agar '!51>"1,,6( !ac.onkey agar '!,=&A"9,6(, :rilliant green agar '!,&139,,6(, JC3 '!! ,1+"1,,6(, )) agar '!1,= 1,,6(, Dektoen agar '!7+>"1,,6(, 6ram negative rods 6ram negative rods

1&

5.

Campylobacters '.ampylobacterisis(

Thiol medium '! =9&"9,,6(, )kirrow agar '83 19= )elective supplement to .ampylobacter agar(, !ac.onkey agar '!,=&A"9,6(, :utzler medium '83,,> supplement( :lood agar '!,&>"1,,6(, :D* agar '!&11"1,,6(, 3)A agar '!1=5"9,,6( :lood agar '!,&>"1,,6(, !ac.onkey agar '!,=&A"9,,6( .hocolate Agar ' !odified from :lood agar(, Cevinthol medium '!7>&"9,,6(, :lood agar '!,&>"1,,6( Albimi medium '!,>7 9,,6(, .olumbia Agar '!/177 9,,6(, 3extrose starch agar '!1=5"9,,6( :lood agar '!,&>"1,,6( !ac.onkey agar '!,=&A"9,6( 00CE Agar '3ifco":3 &997&,(, Ex heart infusion agar '0repared at Caboratory( 2!ND medium, Oorthof medium '!79>"1,,6(, 8letcher1s medium '!&5A"1,,6( !annitol salt agar '!/11="1,,6(, :aird"0arker medium '!2 ,75" 1,,6(, 0urple agar '!altose agar( :lood agar '!,&>"1,,6(

6ram negative rods

7.

Pasteurella multocida '8owl cholera(

:ipolar 6ram negative rod :ipolar 6ram negative rod 6ram negative rod

9.

Mannhermia haemolytica 'Daemorrhagic )epticaemia(

+.

Haemophilus paragallinarum '*nfectious .oryza(

>.

Brucella spp ':rucellosis(

!odified acid fast organisms 6ram negative rod 6ram negative pleomorphic 6ram negative spirochaete 6ram positive cocci in bunches

=. A.

Actinobacillus lignieresi '4ooden tongue( Mycoplasmas spp. '!ycoplasmosis(

1,.

Leptospira sero ars 'Ceptospirosis(

11.

Staphylococcus spp. !astitis, :umbled foot(

15

1&. 15. 17.

Streptococcus spp.'!astitis, )trongyles in 2quine ( Bacillus anthracis Anthrax Clostridium per!ringens '2nterotoxiemia( Clostridium chau oei ':lack quarter( Clostridium tetani 'Tetanus(

2dward1s medium '!>7="1,,6(, :lood agar '!,&>"1,,6( 0C2T agar '!&+A "9,,6(, :lood agar '!,&>"1,,6( .ooked meat medium '!17A" 1,,6(, :lood agar '!,&>"1,,6( .ooked meat medium '!17A"9,,6( :lood agar '!,&>"1,,6(

6ram positive cocci in chains 6ram positive rods 6ram positive rod 6ram positive rod 6ram positive rod 3rumstick bacilli 6ram positive rod 6ram positive rod 6ram positive rod

19. 1+.

1>. 1=.

Listeria monocytogenes 'Cisteriosis( Erysipelothri" rhusiopathiae ')wine erysipelas( Actinomyces bo is 'Cocked?Cumpy #aw(

:lood agar '!,&>"1,,6( )erum agar '0repared at Cab(, :lood agar '!,&>"1,,6( 3orset egg medium ')C ,&9(, Coeffler1s serum '!11=A"1,,6(, :D* agar '!&1, "9,,6(, 6lucose broth '!,>,(, Thioglycollate broth '!,1,"1,,6( 3orset egg medium ')C ,&9(, Cowenstein"Nensen1s medium '!1A=, 83,1A"Di"!edia5B )tonebrinks medium Derrold1s egg"yolk medium

1A.

&,.

Mycobacterium spp 'Tuberculosis(

Acid fast orgnisms

&1.

Mycobacterium Paratuberculosis

Acid fast orgnisms

'Nohne1s disease( Note; .atalogue Bo. starts with ! are Dimedia products and others are 3ifco products.

17

. S2MP8E P9<!ESSIN$ 2ND !>8T>9E '<9 IS<82TI<N <' *2!TE9I2 Sa",le 'processed without delay and inoculated into appropriate media( :rain heart infusion ? nutrient broth 7 to + hours incubation 5>@. *noculation into solid media 'overnight growth( .olonies in different culture media *rilliant $reen agar 6reen colonies I Escherchia coli % 0ink colonies with pink background" Salmonella# :right yellow colonies " 0roteus% Pellow mucoid colonies" Klebsiella pneumoniae Bo growth" Pasteurella )p. Mannitol salt agar C Ed-ards "edi%" Pellow colonies with yellow background I Staphylococcus aureus 4hite colonies" Staphylococcus intermedius Staphylococcus epidermis Enly salt tolerant organism will grow'Dalophilic( Sa)o%ra%ds De7trose agar <apidly growing velvety granular ? bluish green growth I Aspergillus !umigatus :lack and granular growth" Aspergillus niger Pellowish green with fluffy texture I Aspergillus !la us This will inhibit the growth of most of the bacteria in addition .hloramphenicol and .ycloheximide increase selectivity of pathogenic fungi.

*lood agar :eta haemolytic '0artial haemolysis( in Staphylococcus# Dot cold 0henomenon on refrigeration

Mac!on.ey agar Pin. flat colonies 'Cactose fermenting( with bile precipitation" Escherchia coli Pin. !on0e7 colinies without bile precipitation I Enterobactor sp. 0artial pink mucoid colonies" Klebsiella sp. .olourless colonies" )almonella, )higella, 0roteus Bo growth" Pasteurella sp. 'except P.trehalosi(

N%trient agar !edusa head colonies I Bacillus sp. :luish green colour change I Pseudomonas sp.

Bon"haemolytic minute pin point colonies in Pasteurella multocida

This will support many type of organisms 'The colony growth from this agar should be used for catalase and oxidase test(

19

.# *acterial gen%s identification flochart

$ra" D

$ra"s staining 6ram ""Q Exidase K

Campylobacter Helicobacter $ibrio

.atalase

Exidase "" 6rowth on !A.

6rowth K 6rowth ""

6rowth on !A.
.atalase 0ositive .atalase negative

6rowth K
Micrococcus Staphylococcus Bacillus Listeria Corynebacterium Actinomyces% &hodococcus Streptococcus '( ) *+ Enterococcus '(+ Erysipelothri" '(+ A.pyogenes ')+ Nocardia% Actinobacillus 'pin,+ Aeromonas 'PL-+ Bordetella 'Clear+ Pseudomonas 'Clear+ Acinetobacter

6rowth ""
Brucella Haemophilus Mora"ella Pasteurella

Cactose K?""
Brucella o is

*f R )taph .oagulase S !)A, !altose agar

*f rods !otile

*f T )trep, :ile )alt 2sculin hydrolysis

*f R )trep, 0atho"
3x Catex Test kit

Citrobacter Enterobacter Escherchia Klebsiella

.ersinia Proteus Salmonella Serratia

Bacillus Listeria )et up )elenite :roth, :rilliant 6reen Agar ':6A(, !A., Jylose Cysine 3eoxycholate, D2A

:lack colonies on Dektoen enteric Agar 'D2A( ,Bon Cactose fermenting 'BC8( on !ac .onkey

Tiple )ugar *ron Agar S .itrate

0ink colonies )elenite broth subculture on :6A

T)* 'Alk?D&)

8ecal sample for Salmonella

Bo :lack colonies on D2A

6rouping by )almonella antiserum

1+

.4 Strea.ing "ethods and isolation of ,%re c%lt%re !o""on Methods of isolation of ,%re c%lt%re 0ure culture of microorganisms that form discrete colonies on solid media, e.g., yeasts, most bacteria, many other fungi, and unicelluar microalgae, may be most commonly obtained by plating methods such as Strea. ,late "ethodB Po%r ,late "ethod and S,read ,late "ethod. :ut, the microbes that have not yet been successfully cultivated on solid media and are cultivable only in liquid media are generally isolated by serial dilution method. Strea. Plate Method This method is used most commonly to isolate pure cultures of bacteria. A small amount of mixed culture is placed on the tip of an inoculation loop?needle and is streaked across the surface of the agar medium. The successive streaks $thin out$ the inoculum sufficiently and the microorganisms are separated from each other. *t is usually advisable to streak out a second plate by the same loop?needle without re"inoculation. These plates are incubated to allow the growth of colonies. The key principle of this method is that, by streaking, a dilution gradient is established across the face of the 0etri plate as bacterial cells are deposited on the agar surface. :ecause of this dilution gradient, confluent growth does not take place on that part of the medium where few bacterial cells are deposited =ario%s "ethods of strea.ing

')ource " http ??www.microbiologyprocedure.com( 0resumably, each colony is the progeny of a single microbial cell thus representing a clone of pure culture. )uch isolated colonies are picked up separately using sterile inoculating loop? needle and re"streaked onto fresh media to ensure purity.

1>

Po%r Plate Method This method involves plating of diluted samples mixed with molten agar medium. The main principle is to dilute '1?1,( the inoculum by transferring 1ml of specimen culture to A ml of liquid media, after proper mixing, 1ml will be transferred to the subsequent tube( so as to permit a thorough distribution of bacterial cells within the medium. Dere, the mixed culture of bacteria is diluted directly in tubes containing molten agar medium maintained in the liquid state at a temperature of 7&"79-. 'agar solidifies below 7&-.(. The contents of each tube are poured into separate petri plates, allowed to solidify, and then incubated. 4hen bacterial colonies develop, one finds that isolated colonies develop both within the agar medium 'subsurface colonies( and on the medium 'surface colonies(. These isolated colonies are then picked up by inoculation loop and streaked onto another 0etri plate to insure purity. 0our plate method has certain disadvantages as follows 'i( the picking up of subsurface colonies needs digging them out of the agar medium thus interfering with other colonies, and 'ii the microbes being isolated must be able to withstand temporary exposure to the 7&"79temperature of the liquid agar medium% therefore this technique proves unsuitable for the isolation of psychrophilic microorganisms. Dowever, the pour plate method, in addition to its use in isolating pure cultures, is also used for determining the number of viable bacterial cells present in a culture. Diagra""atic re,resentation of Po%r Plate Method 2. !edia?dilution *. 0ouring of the !. .olony development after incubation. .ontrol plate% and consists of the sterilized plating medium alone

')ource " http ??www.microbiologyprocedure.com(

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The isolated colonies are picked up and transferred onto fresh medium to ensure purity. *n contrast to pour plate method, only surface colonies develop in this method and the microorganisms are not required to withstand the temperature of the molten agar medium.

.& !<8<NE M<9P:<8<$E

Mac!on.ey agar; 8'CN8' colony

:ae"olysis on )lood agar

8ecitheniase acti0ity on egg yol. agar

!oli for" co%nt agar FPin. coli for"

Pseudomonas aerogenosa "%coid colonies

E.coli Metallic sheen on EM* agar

Staph s,. on Mannital salt agar

Med%sa head - Bacillus colony

1A

Listeria on P28!2M agar

Pseudomonas ; chro"ogenic colony

Salmonella on X8D

Proteus-S-ar"ing on )lood agar

Vibrio colonies on T!*S agar

Staph.on *aird ,ar.er agar

So%rce ; http ??www.microbelibrary.org?A)!Enly?details.asp

S,read ,late "ethod

')ource " http ??www.microbiologyprocedure.com(

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The inoculum is sub#ected to serial dilution in a sterile liquid medium, and a large number of tubes of sterile liquid medium are inoculated with aliquots of each successive dilution and spread over the plate. 0lates are incubated and observed for bacterial growth.

.( Maintenance and Preser0ation of P%re !%lt%res

Ence a microorganism has been isolated and grown in pure culture, it becomes necessary to maintain the viability and purity of the microorganism by keeping the pure cultures free from contamination. Bormally in laboratories, the pure cultures are transferred periodically onto or into a fresh medium 'sub"culturing( to allow continuous growth and viability of microorganisms in slant. The transfer is always sub#ect to aseptic conditions to avoid contamination. 2gar slant Pre,aration Agar slants are prepared to inoculate microbial culture. To prepare agar slant a required number of culture tubes are taken and about 9 I1, ml of liquefied agar medium is poured in each of them.

')ource " http ??www.microbiologyprocedure.com( The tubes in are an cotton"plugged autoclave. and After

sterilized

sterilization the tubes are taken out and are placed in slanting 'sloping( position for sometimes% the tubes get cooled and the medium in them is solidified resulting in a slopy surface.

&1

Inoc%lation Proced%re 1. *mmediately after deplugging and sterilizing the mouth of the tubes, the loop?needle is also flame"sterilized and is inserted within the agar surface of the inoculum containing tube to quench the heat, and a small bit of inoculum is taken on the loop?needle tip. &. The inoculum containing loop?needle is taken out and brought in within the agar slant containing tube where the inoculum is #ust rubbed on the surface of the agar slant. 5. All the steps starting from plug removal from the mouth of the tubes to the rubbing of the inoculum on the surface of the agar slant should be done quickly to avoid contamination. 7 4hen inoculation is complete, the open mouths of tubes and the cotton plugs are sterilized by flame and the cotton plugs are replaced. 9. The inoculated tube is incubated under suitable temperature to favour rapid growth of microorganisms. )ince repeated sub culturing is time consuming, it becomes difficult to maintain a large number of pure cultures successfully for a long time. *n addition, there is a risk of genetic changes as well as contamination. Therefore, it is now being replaced by some modern methods that do not need frequent subculturing. These methods include refrigeration, paraffin method, cryopreservation, and lyophilization 'freeze drying(. 9efrigeration 0ure cultures can be successfully stored at ,"7-. either in refrigerators or in cold" rooms. This method is applied for short duration '&"5 weeks for bacteria and 5"7 months for fungi( because the metabolic activities of the microorganisms are greatly slowed down but not stopped. Thus their growth continues slowly, nutrients are utilized and waste products released in medium. This results in, finally, the death of the microbes after sometime. Paraffin Method This is a simple and most economical method of maintaining pure cultures of bacteria and fungi. *n this method, sterile liquid paraffin in poured over the slant 'slope( of culture and stored upright at room temperature. The layer of paraffin ensures anaerobic conditions and prevents dehydration of the medium. This condition helps microorganisms or pure culture to remain in a dormant state and, therefore, the culture is preserved for several years.

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$lycerol Preser0ation; 6lycerol prepared in 1,F v?v with autoclaved :D* broth could be used as preservative and maintenance medium for bacterial culture. After inoculation of culture the medium is incubated for 7hours and then kept in "&,. for as long as &years. At the time of requirement the glycerol culture is revived by sub culturing in a suitable medium. !ryo,reser0ation .ryopreservation 'i.e., freezing in liquid nitrogen at "1A+-.( helps survival of pure cultures for long storage times. *n this method, the microorganisms of culture are rapidly frozen in liquid nitrogen at "1A+-. in the presence of stabilizing agents such as glycerol that prevent the formation of ice crystals and promote cell survival. 8yo,hilization '8reeze"3rying( *n this method, the culture is rapidly frozen at a very low temperature '">,-.( and then dehydrated by vacuum. /nder these conditions, the microbial cells are dehydrated and their metabolic activities are stopped% as a result, the microbes go into dormant state and retain viability for years. Cyophilized or freeze"dried pure cultures and then sealed and stored in the dark at 7-. in refrigerators. 8reeze"drying method is the most frequently used technique by culture collection centres. #. ST2ININ$ *2!TE9I2 #.1 Pre,aration of !lean slide; *t is essential to use clean, dry, dust"free slides remember that grease and residual detergent are equally liable to spoil a blood film and interfere with staining. Ne- slides; :oxes of clean grease"free slides may be used. *f not, available proceed as follows. Ceave the slide overnight in a detergent solution. Then wash thoroughly in running tap"water, rinse in distilled water if available and wipe dry with a clean linen cloth. :efore use, wipe the surface with methylated spirits 'A9F ethanol( or methanol and dry with a clean cloth% then keep covered to avoid having dust settle on the surface. >sed slides; 3iscard in detergent solution, heat to about +,o. for &, minutes. Then wash in running tap"water, rinse in distilled water if available and treat as for new slides as described above.

&5

#. $ra" Stain;
6rams staining reaction classify bacteria broadly in to 6ram positive and 6ram negative. :acteria that decolorize easily are called 6ram"negative '0ink?red( and those that retain the primary stain are called 6ram"positive 'purple(. :acteria stain differently because of differences in their cell walls. 6ram"positive cell walls consist of many layers of peptidoglycan. The crystal violet"iodine complex is larger than either the crystal violet or iodine molecules that entered the cell and the complex cannot pass through this thick cell wall. 6ram"negative bacteria have a thin layer of peptidoglycan and an outer lipopolysaccharide layer. The alcohol dissolves the lipopolysaccharides so that the crystal violet"iodine complex can be washed out of the cell. :efore bacteria can be stained, a smear of bacteria must be made on a slide and heat fixed. A smear is made by spreading a bacterial suspension on a clean slide and letting it air dry. The dry smear is heated on a hot plate or passed through a flame several times to heat fix it. Pre,are a )acterial s"ear.

0lace 1, Hl of sterile water in the center of a clean glass slide. !ix bacteria with water drop on slide. )pread the water"bacteria mixture over an area of about 1 inch square. Allow the smear to air dry. &7

Dold the slides with forceps and heat fix the smears by heating on the hot plate for several minutes.

Staining Proced%re; .over the smear with a few drops of crystal violet and leave for 1 minute. Turn the slide over so the smear is facing down. 4ash the slide carefully over the top with distilled water from a wash bottle over the sink until no large amounts of color wash off. 3o not squirt water directly onto the smear. .over the smear with 6ram1s iodine and leave for 1 minute. 4ithout washing, decolorize with A9F ethyl alcohol '3ecolorizer 4ash the slide carefully over the top with distilled water .over the smear with safranin and leave for 1 minute. 6ently rinse with distilled water. :lot dry with paper towel or absorbent paper. 3o not rub slide, as the bacterial smear can be rubbed off. Ebserve under microscope. <ecord the observations from the microscope.

#.# 2lternati0e test for $ra"s reaction - # G Potassi%" :ydro7ide a. Add heavy inoculum of pure culture of bacteria grown on solid medium to a drop of 5 F potassium hydroxide 'OED( solution '5 grams OED per 1,, mC distilled water( on a clean glass slide ). )tir for about one minute, occasionally lifting the loop to look for thickening and GstringingU of the slurry <)ser0ation; 1. 6ram positive bacteria will not appear to change the viscosity of the OED solution &. 6ram negative bacteria will cause the OED solution to become stringy or mucoid in appearance and consistency. 5.7 3iehl-Neelsen 43N5 staining The high mycolic acid content of certain bacterial cell walls, like those of Mycobacteria, is responsible for the staining pattern of poor absorption followed by high retention. The most common staining technique used to identify acid"fast bacteria is the Viehl"Beelsen stain, in

&9

which the acid fast bacilli are stained bright red and stand out clearly against a blue background 2g Proced%re ; .over the smear with carbol"fuchsin solution and heat under the slide with spirit lamp to make the carbolfuchsin pierce the cell wall mycolic acid components.' )lide should not get dried( Allow the slide to stand in hot solution for 9 minutes. 4ash with running tap water. 3ecolorize with 1F Acid alcohol until light pink color appear. 4ash in running tap water for 9 minutes. .over the smear with methylene blue for 5, seconds. <inse in water. Air dry the slide and observe under microscope All !ycobacteria " M. tuberculosis, M. leprae, M. smegmatis and atypical Mycobacterium , Nocardia

#.& Staining techni6%e for 2nthra7 )acilli <)Hecti0e !icroscopic examination of clinical material for anthrax bacilli to establish a Wsuspect1 diagnosis of anthrax and of environmental materials for the presence of anthrax spores. Pre,aration of s"ears; !ake two thin smears of clinical?animal material by rolling over the swabs or spreading a small drop on a microscope slide Air dry and fix by dipping the slide in absolute alcohol for 5,I+, seconds. )lide should not be heat dried to avoid distortion of morphology of the capsule.

<)ser0e the ty,ical "or,hology of the )acill%s; *n clinical material B. anthracis are 6ram positive thick, short, straight bacilli with square or truncated ends with parallel sides found usually single, in pairs or chains of three or four bacilli. The chain of bacilli with truncated and swollen ends gives a characteristic $bamboo stick$ appearance. Polychro"e "ethylene )l%e stain for ca,s%le 4MA'adyean reaction5 This is the ideal method for demonstration of the capsule. 0ut a large drop of polychrome methylene blue on the smear to cover it completely. Ceave for 5,I+, seconds and air dry. 4ash off 'into hypochlorite solution, 1,,,,, ppm(. A wash bottle is better than a tap. 2xamine under oil immersion. &+

The capsule is seen clearly as pink amorphous material surrounding the blue"black bacilli.

<ther staining techni6%es; Ceishman and 6iemsa stains are recommended for staining .erebrospinal fluid '.)8(, other body fluids and blood, as the organisms are clearly visualized from the cellular background. 8eish"an stain 0our =I1& drops of Ceishman stain on the smear. 4ait for two minutes. 3ilute with double the volume of buffered distilled water 'pD ad#usted to >.&(. !ix by rocking the slide or with a 0asteur pipette. Oeep for >I1, minutes. <un tap water on to the slide to float off the stain. 3rain and dry vertically in air. 2xamine under oil immersion.

The bacilli stain purple with a clear space around indicating capsule $ie"sa stain This is used especially for staining blood smears. 0repare fresh 6iemsa stain by diluting stock stain 1 1, in buffered distilled water 'pD ad#usted to >.&(. Air dry films and fix in methanol for one minute 4ash off with wash bottle into hypochlorite solution. 8lood the slide with 6iemsa stain and stain for &9I5, minutes. <un tap water on to the slide to float off the stain and to prevent deposition of precipitate on to the film. 3rain, dry vertically and examine under oil immersion. The bacilli stain purple with red capsule. 8eish"an $ie"sa staining Pre,aration of 8eish"an $ie"sa stain-stoc. sol%tion 1. &9, ml of glycerine is heated to +,. in a light proof container '1litre flask covered with black or brown paper(. &. 5.=g of 6iemsa powder and 1 gm of Ceishman powder is added to it and held at +,. for 1 hour under constant stirring. &>

5. &9, ml of acetone free methyl alcohol is added after bringing the above solution to room temperature. 7. The solution is then filtered using 4hatman Bo.1filter paper. The filterate is then collected in an amber coloured bottle and used as stock solution. Staining ,roced%re The smear is fixed by immersing the slide in methanol for one minute. 1. &. 5. Ene drop of the stock solution is added to one ml of tapwater. )mear is then flooded with the diluted stain. *t is allowed to act for 79 min. The slide is then washed in tap water. The slide is air dried and examined under oil immersion of microscope.

4. SE!<ND29E 8E=E8 IDENTI'I!2TI<N <' *2!TE9I2 *E *I<!:EMI!28 TESTS

!ost bacteria are identified and classified largely on the basis of their reactions in a series of biochemical tests. )ome tests are used routinely for many groups of bacteria 'eg. oxidase, nitrate reduction(% others are restricted to a single family, genus, or species 'eg. coagulase test for staphylococci(. 4.1 *ioche"ical Proced%res and o)ser0ation 1. !atalase Test .atalase test is used to detect the production of catalase enzyme by the bacterium. .atalase enzyme is produced by most bacteria. The enzyme break hydrogen peroxide 'D&E&( and release free Exygen. .atalase is found in most aerobic and facultative anaerobic bacteria. The main exception is Streptococcus spp. That1s why the test is used to differentiate between Staphylococcus and Streptococcus. 3ip a capillary tube into 5F D&E& and touch a bacterial colony Ebserve the tube for bubble production :ubbling indicates a positive result. . <7idase The oxidase test identifies certain organisms that produce the enzyme cytochrome oxidase. .ytochrome oxidase participates in the electron transport chain by transferring electrons from a donor molecule to oxygen. The oxidase reagent contains a compound that changes color when it becomes oxidized. *f the test organism produces cytochrome oxidase, the colorless reagent used in the test will detect the presence of the enzyme oxidase and, reacting with oxygen, turn violet to purple. The oxidase test is a key test to differentiate &=

between the families of 0seudomonadaceae 'oxidase positive( and 2nterobacteriaceae 'oxidase negative(. The test reagent is smeared on a filter paper or readymade reagent discs The target single colony is picked up in a non reacting material like glass rod or plastic stick and then streaked on the disc 3evelopment of Miolet colour within 19 "5,seconds is considered as positive :acteria metabolize carbohydrates by oxidative and?or fermentative pathways. Exidation occurs in the presence of atmospheric oxygen 'aerobic(, whereas fermentation takes place in an anaerobic environment. !etabolism of the carbohydrate dextrose by either an aerobic or anaerobic pathway results in acid production. The resulting acidic environment causes the :romo Thymol :lue pD indicator in the medium to turn from green to yellow. The presence of bubbles in the tube indicates gas production 'aerogenic(. *f no reaction occurs, the medium can remain unchanged or become alkaline 'blue at the surface(. A deep butt tube 'X> mC in 1+ Y 1&9mm( is used for this test. 4ith a sterile needle take a small inoculums from an isolated colony and stab to the bottom of the tube. *ncubate at &,"&7-. for &7"7= hours. .heck tubes at &7 hr for acid and?or gas production. 9ES>8TS 9eaction Exidative 8ermentative Bon"reactive 4. Tri,le S%gar Iron 4TSI5 This medium can determine the ability of an organism to utilize specific carbohydrates incorporated in a basal growth medium, with or without the production of gas, along with the determination of hydrogen sulfide 'D&)( production. T)* agar contains the three sugars in varying concentrations glucose '1J(, lactose '1,J(, and sucrose '1,J(. *t also contains the pD indicator phenol red. *f sugar fermentation occurs, glucose will be initially used and the butt of the tube will be acidic 'yellow(. After glucose utilization the organism may continue to ferment the remaining sugars. *f this occurs the entire tube will become acidic. .ertain bacteria are unable to utilize any sugars and will breakdown the peptone present. 0eptone &A A Z acid'yellow(% A6 Z acid K gas, B Z no change or alkaline To, of T%)e A A6 or A B *otto" of T%)e B A6 or A B #. $l%cose 'er"entation 4<' *asal -ith 1G $l%cose5 F

utilization causes an alkaline 'red( shift in the medium that causes a color change from orange to red. :lackened medium is caused by hydrogen sulfide production, which changes ferrous sulfate to ferrous sulfide. *n addition, splitting of the medium or presence of bubbles in the butt of the tube can determine gas production. 4ith a sterile needle inoculate the T)* slant by stabbing to the bottom of the tube and then streaking the surface of the slant as the needle is drawn out of the tube. *ncubate with loosened cap. <ead the results after 1="&7 hours. 9ES>8TS; A Z Acid% O Z Alkaline% D&) Z Dydrogen sulfide produced% B ZBo change SlantC*%tt O?B or O?A !olorC9eaction <ed? Erange 'Exidative( or Inter,retation red ?yellow Enly peptone utilized or only glucose"fermented 6lucose, plus lactose 6as production Dydrogen sulfide produced

'fermentative( A?A Pellow?Pellow and ?or sucrose Ifermented 6as )plitting or bubbles D&) :lack butt &. !ar)ohydrate >tilization

The following carbohydrates are normally used in laboratory to aid in bacterial species identification Arabinose, <hamnose, !annitol, )alicin, )orbitol, and )ucrose 'saccharose(. The procedures to be followed for each of these media are identical. up to four days may be necessary for some negative results. 9ES>8TS a( 0ositive " Acid is produced from fermentation, which turns media yellow. b( Begative " Bo fermentation of carbohydrate, media remains same. c( Aerogenic " 6as bubbles are present within the media. (. <C1 1 Discs This test determines the sensitivity of a bacterial organism to the vibriostatic agent &,7"diamino"+,> di"isopropylpteridine 'E?1&A(. )uspend bacteria in sterile saline or 0:) )treak suspension on plate in three planes with a cotton swab. Aseptically place sensitivity disc in the center of inoculum. 5, *noculate carbohydrate tube with a loop full of 1= to &7 hour pure culture. *ncubate with loosened cap for 1= to &7 hours at 5>o.. A prolonged incubation of

*ncubate at &,"&7-. for &7 hours. This test can be done on the same plate as the antibiotic sensitivity test. )ensitive Vone of inhibition around disc <esistant 6rowth ad#acent to disc

9ES>8TS;

+. IM=i! Tests The *!Mi. series includes four tests A. *ndole production :. !ethyl red test .. Moges"0roskauer test 3..itrate utilization 2xcept for the lowercase GiU, which is added for ease of pronunciation, each of the letters in G*!Mi.U stands for one of these tests. G*U is for indole% G!U is for methyl red% GMU is for Moges"0roskauer, and G.U is for citrate. *!Mi. tests are particularly useful for differentiating Escherichia coli and Klebsiella pneumoniae 2. Indole ,rod%ction The ability to degrade amino acids to identifiable end products is often used to differentiate among bacteria. )ome bacteria can hydrolyze tryptophan using an enzyme called tryptophanase. Carge amounts of energy can be obtained by this hydrolysis. A byproduct of the hydrolysis is indole, which is excreted by the organism. *ndole can be detected by reaction with Oovac;s reagent 'para"dimethylaminobenzaldehyde in alcohol( which produce a red color. A red layer indicates a positive result. *t is used mainly in the differentiation of genera and species. 8or example# E. coli gives a positive result while Proteus mirabilis and Klebsiella give a negative one. *ndole production is dependent upon the presence of tryptophan in the culture medium. A 1F solution of tryptone is generally used in tests for indole production because it is rich in tryptophan. :acteria to be tested are incubated for 7=">& hours. 9 drops of Oovac;s reagent are added to the culture broth. *ndole reacts with Oovac;s reagent to give a red product in the alcoholic layer. A yellow color indicates a negative result. *. Methyl 9ed and !. =oges-Pros.a%er Tests 4M9-=P5 The fermentation of glucose by bacteria results in end products that vary from species to species depending on metabolic pathways that are available to them under the culture conditions. A number of genera of 6ram negative bacteria ferment glucose to produce lactic, acetic, succinic, and formic acids 'mixed acid fermentation(. They also typically produce large amounts of .E&, D&, and ethanol. Acid accumulation can reduce the pD to 9 or lower. Ether organisms ferment glucose to form only one fermentation product, usually acetic acid 51

which is converted to acetoin. !< test 'methyl red( is used to determine if glucose can be converted to acidic products like lactate, acetate, and formate, acid products that will cause the pD to drop below 7. !ethyl red, an indicator, turns red below 7.7 and will thus turn red if acid products are present, otherwise it will remain yellow. M0 'Moges"0roskauer( test is used to determine if glucose can be converted to acetoin. :arritt1s <eagent A and : are added. The reagents will react with acetoin, and a positive reaction will show a red color. Proced%re; Methyl red test; 1. *noculate the organism and incubate it for 7= hrs at 5>@.. &. Add a few drops of methyl red to the original culture tube and mix the contents. 9es%lts ;A red color indicates a positive result. A yellow color indicates a negative result. !. =oges-Pros.a%er test; 1. *noculate the organism and incubate it for 7= hrs at 5>.. &. Add of :arritt;s reagent A to the tube and mix. 5. Add 19 drops of :arritt;s reagent :. and mix. 7. The culture should be kept on table for about &, minutes for color development to occur. 9. A red color indicates a positive result. A yellow color indicates a negative result. D. !itrate >tilization )ome bacteria may be able to use organic compounds other than sugars as their sole source of carbon. The citrate test determines if a bacterium can grow utilizing citrate as its sole carbon and energy source. These bacteria are able to cleave citrate to oxaloacetate and acetate via the citritase enzyme. Si""onsCIoserJs citrate "edi%" This medium is used to test for citrate utilization. Ooser .itrate !edium is prepared so that no sources of carbon 'other than sodium citrate( or nitrogen 'other than ammonium salts( are present. :acteria that are able to use citrate as their carbon source will grow in the medium and change the green coloured medium to blue colour change which indicates a positive reaction. Proced%re; 1. Transfer growth from a single colony or a loopful of liquid suspension inoculate the .intrated slant medium. &. *ncubate at 5>@ . for 1="&7 hours. 5. The development of blue colour indicates a positive result while a clear green colour is 5& and

considered as negative. The results of the *!Mi. Test can be used to differentiate between E.coli and Klebsiella. 2.coli gives KK"" results on the *!Mi. tests, while Klebsiella give the reverse "" KK . /. !oag%lase Test The test is used to detect the production of the enzyme coagulase which converts fibrinogen to fibrin. *n the laboratory, it is used to distinguish between different types of Staphylococcus isolates '.oagulase 0ositive staphylococcus '.0)( or .oagulase Begative )taphylococcus '.B)(. .oagulase reacts with prothrombin in the blood. The resulting complex causes blood to clot by converting fibrinogen to fibrin. The test is perfomed as slide .oagulase test 'for bound .oagulase( or tube .oagulase test 'for free .oagulase( A suspension of )taphylococcus culture is mixed with rabbit plasma either on a glass slide or in small tube. 8ormation of clump in slide or gelling and clot formation of rabbit plasma in tube is positive. *n tube test time is noted as .oagulase positive in & hrs, 7hrs, +hrs etc. )lide test is presumptive and tube test is confirmative coagulase test. /rease is an enzyme that catalyzes the hydrolysis of urea into carbon dioxide and ammonia. The reaction occurs as follows 'BD&(&.E K D&E [ .E& K &BD5 /rease broth is a differential medium that tests the ability of an organism to produce urease. The ready made urea broth contains two pD buffers, urea, a very small amount of nutrients for the bacteria, and phenol red as pD indicator. /rea broth should not be autoclaved but filter sterilization is required before inoculation. !embers of the genus Proteus and Bordetella can degrade urea rapidly. The test is used to differentiate urease"positive Proteus species from others members of the 2nterobacteriaceae. )ome strains of Klebsiella species are also urease positive. 9es%lts ; 0henol red turns yellow in an acidic environment and fuchsia in an alkaline environment. *f the urea in the broth is degraded and ammonia is produced, an alkaline environment is created, and the media turns pink.

1. >rease

55

10. Nitrate red%ction test The identification of some bacteria is aided by determining if the organism can reduce nitrate 'BE5( to nitrite 'BE&( or another nitrogenous compound such as ammonia 'BD5( or nitrogen gas 'B&(. This reaction is expressed as BE5 """"\ BE& """"\ BD5 or B& *n order to determine if a bacteria can reduce nitrate, the test organism is inoculated into nitrate reduction broth. Alpha"napthylamine and sulfanilic acid are added and if a red color develops then the bacteria is capable of reducing nitrate to nitrite. *f the bacteria converts nitrate to nitrogen gas this can be detected using a 3urham1s tube which will be elevated if gas is evolved. Bitrate positive bacteria include E. coli/ Salmonella/ Klebsiella pneumoniae. Enterococcus !aecalis/ Bacillus subtilis. 11. Decar)o7ylase Test 48ysine and <rnithine5 A determination of bacterial enzymatic capability to decarboxylate an amino acid to form an amine with resultant alkalinity. 8or each isolate to be tested, it is necessary to inoculate a decarboxylase control tube and lysine or ornithine test tube. /se very small amount of inoculum from 1= to &7 hour pure culture. Add 1 to & mC oil overlay to each tube. *ncubate at 5>. for &7 hours )ome time a prolonged incubation of up to four days may be necessary. 8ysine or <rnithine T%)e Turbid to faded purple 'glucose fermented, decarboxylase produced( Pellow 'glucose fermented, decarboxylase not produced( 0urple 'glucose not fermented, decarboxylase not produced( !ontrol T%)e Pellow 'glucose fermented( Pellow 'glucose fermented( 0urple 'glucose fermented( not

9ES>8TS Test 9es%lt 0ositive Begative Begative

1 . Esc%lin Test To determine the ability of an organism to hydrolyze the glycoside esculin 'aesculin( to esculetin 'aesculetin( and glucose in the presence of bile '1, to 7,F(. *noculate the surface of the bile esculin slant with inoculum from an 1= to &7 hour old pure culture. 57

 *ncubate at 5>-. for &7 to 7= hours. 9ES>8TS a( 0ositive " 0resence of a black to dark brown color on the slant. b( Begative " Bo blackening of the medium. P9E!2>TI<NS 8alse positives may occur with hydrogen sulfide producing organisms. Beither of the target organisms for these protocols will, however, produce hydrogen sulfide. 1#. $elatinase A test to determine bacterial production of gelatinase enzymes that liquefy gelatin. *noculate by stabbing ] to 1 inch deep into the nutrient gelatin media with a heavy inoculum from an 1= to &7 hour pure culture. *ncubate 1= to &7 hours at &,"&7-.. 9ES>8TS a( 0ositive I !edia is liquefied. 4eak results can be visualized by rapping the tube against the palm of the hand to dislodge droplets of liquid from the media. Any drops seen are considered positive. b( Begative I Bo liquefaction occurs in media. 14. Motility This test determines if a bacterial isolate is motile by means of flagella. 0lace a drop of distilled water or sterile 0:) onto the center of a clean microscope cover glass. 0lace an additional tiny drop in one corner of the cover glass 'to adhere the cover glass to the depression slide when it is inverted(. *noculate the center drop from a pure culture that is &7"7= hours old using a sterile loop, however +"=hrs culture will show actively motile bacterium. .arefully invert the cover glass and place over the concave portion of a hanging drop slide. Ebserve for motility using 7,,Y magnification on a compound microscope. .are should be taken to not interpret GdriftU or G:rownian motionU as motility. <ecord results as motile or non" motile. If this "ethod fails to sho- "otility then; a( *noculate a nutrient broth with the isolate and incubate at room temperature until growth is obtained, usually &7 hours. After incubation use a sterile loop or sterile dropper and place a drop on a clean cover glass. 0lace a tiny drop of distilled water in one corner of the same cover glass. .ontinue as above. 59

b( )emi"solid motility test medium can also be used. )tab the medium with a small amount of inoculum. *ncubate overnight at room temperature. *f the bacterial species is motile, the medium will become turbid with growth that radiates from the line of inoculum. *f the visible bacterial growth. bacterial species is non"motile, only the stab line will have

4. M2K<9 $92M NE$2TI=E *2!TE9I2 <' =ETE9IN29E IMP<9T2N!E 2ND ITS *I<!:EMI!28 9E2!TI<NS
<9$2NISMS Escherichia Klebsiella Enterobacter Citrobacter Salmonella Shigella Proteus Aeromonas Pro idencia Ed0ardsiella Serratia Pseudomonas Acinetobacter Pasteurella hemolytic Pasteurella multocida Pasteurella multocida '0oultry( Pasteurella Pneumontropic a Actinobacillus .ersina Bordetella Mora"ella Brucella TSI : S IND<8E >9E2 <XID2SE !IT92TE M<TI8ITE A?A " K " " " K A?A " " K " K " ACO?A " " " " K K ACO?A K K K " K K ACO?A K " " " K K ACO?A " K " " " " ACO?A K K " " K KKK A?A " K " K K K ACO?A " K " " K K ACO?A K K " " " K ACO?A " " " " K K ACO?B. " " " K K K ACO?B.?ACO " " " K K " A?A " " " K " " A?A A?A A?A A?A ACO?A ACO?B. ACO?B. ACO?B. " " " " " " " K K " K " " " " " " " K K K KKK " KK K K K K " K K K " " " " " K K " " " " " K K " "

5+

4.# M2K<9 $92M P<SITI=E *2!TE9I2 <' =ETE9IN29E IMP<9T2N!E 2ND ITS *I<!:EMI!28 DI''E9ENTI2TIIN$ '2!T<9S
hae"olysis Mannital !oag%lase $elatin DN2SEC 2l.aline ,hos,hatase Maltose C P%r,le agar 2esc%lin hydrolysis S%gar fer"entation 8a) ani"al Motility Nitrate 8actose <9$2NISMS 8ecithinase >rease !2MP K K " " K K 60 die <, 60 die <,60 survive

Staph. aureus Staph. 1ntermedius Staph. epidermidis Streptococcus pyogenes Strep. agalactia Strep.dysagalactia Strep.2beris Coryne. Pseudotuberculosis Coryne. &enale. Coryne. o is &hodococcus e3ui Mycobacterium tuberculosis Mycobacterium bo is Mycobacterium a ium

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Listeria monocytogene Erysipelothri" rhusiopathiae Bacillus anthracis K

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4.4 *I<!:EMI!28 TESTS

!ar)ohydrate %tilization test

!atalase test

<7idase test

TSI Slant

>rease test

$elatin 8i6%ifaction

Indole test

Methyl red test

!itrate %tilization test

So%rce ;http ??users.stlcc.edu?kkiser?biochem.html 5A

(. 2NTIMI!9<*I28 S>S!EPTI*I8ITE TESTIN$ Introd%ction Antimicrobial susceptibility tests measure the ability of an antibiotic or other antimicrobial agent to inhibit bacterial growth in itro. This ability may be estimated by either the dilution method or the diffusion method. The recommended method for intermediate and peripheral laboratories is the modified Oirby":auer method, the methodology of which is given below (.1 $eneral g%idelines for ro%tine s%sce,ti)ility testing )usceptibility tests should be carried out only on pure cultures of organisms considered to be causing the infectious process The selection of antibiotic discs depends on the clinical consideration including the drugs that are available and in general use by the veterinarian. A tetracycline disc will predict the results against all other tetracyclines. )ulphisoxazole is a suitable representative for all the sulphonamides Aminoglycosides and quinolones should be tested separately 0encillins, )treptococci should be tested against either pencillin 6 or ampicillin. Testing against both is not necessary. 2rythromycin will predict the result of all other macrolides A clindamycin disc will predict the results for lincomycin. .hloramphenicol, vancomycin, nitrofurantoin and trimethoprim?sulphamethoxazole are tested separately as required. A cephalothin disc will indicate the results for other first"generation cephalosporin1s 'cefalexin, cefradine, cefaloridine, cefazolin, cefapirin(. .efatozime represents ceftazidime, ceftizoxime and ceftriaxone. Modified Iir)y-*a%er "ethod Materials re6%ired Tryptic soy broth or sterile saline broth !ueller"Dinton agar Antibiotic discs o Any commercially available discs can be used and the stocks of antibiotic discs preferably should be kept at "&,@.. 7,

o A small amount of discs can be kept in the refrigerator for up to one month before starting the work discs should be left at room temperature for about one hour to allow the temperature to equilibrate. )terile swabs 3isc dispenser or forceps Proced%re *f the sample provided is milk, then the sample could be directly smeared on agar plate with a sterile swab. *ncase of 0us swab, heart :lood swab or any organ, the sample should be cultured on standard nutrient or :D* agar medium. After standard incubation period, pick 5"9 colonies of similar appearance and transfer to :D* broth or tryptic soy broth. *ncubate the broth cultures at 59 K & o. for &"7 hours for development of light to moderate turbidity or ad#ust the turbidity with ,.9 !c8arland standards. *noculate the plates by dipping a sterile swab into the inoculum. <emove excess inoculum by pressing and rotating the swabs firmly against the side of the tube above the level of the liquid. )treak the swab all over the surface of the medium three times, rotating the plate through an angle of +,o after each application. 8inally, pass the swab round the edge of the agar surface. Ceave the inoculum to dry for a few minutes at room temperature with the lid closed. Apply the antibiotic discs to the inoculated plates using an antibiotic disc dispenser or sterile forceps. The plates should be placed in an incubator at 59 " 5>o. within 5, minutes of preparation and incubated for 1+"1= hours '&7 hours for )taphylococci(. Temperatures above 59o. invalidate the results for oxacillin? methicillin. 3o not incubate in an atmosphere of carbon dioxide. (. Inter,retation After overnight incubation, the diameter of each zone 'including the diameter of the disc( should be measured and recorded in mm. The results should then be interpreted according to the critical diameters by comparing with standard tables as susceptible, intermediate or resistant to the antimicrobial agents used.

71

 The end"point of inhibition is #udged by the naked eye but the extent of zone is complicated in the following situations. Carge colonies growing within an otherwise clear zone of inhibition should be subcultured, identified and retested. 8aint growth of tiny colonies at the edge of the zone can be ignored. 4ith sulfonamides and co"trimoxazole, slight growth occurs within the inhibition zone% such growth should be ignored.

4hen b"lactamase producing staphylococci are tested against penicillin, zones of inhibition are produced with a heaped"up, clearly defined edge% these are readily recognizable when compared with the sensitive control and regardless of the size of the zone of inhibition, they should be reported as resistant.

.ertain Proteus spp. may swarm into the area of inhibition around some antibiotics, but the zone of inhibition is usually clearly outlined and the thin layer of swarming growth should be ignored. 8or bacteria grown on blood agar plates, the zone size for nafcillin, novobiocin, oxacillin and methicillin will be &"5mm smaller than the normal control limits. $eneral g%idelines for ro%tine s%sce,ti)ility testing )usceptibility tests should be carried out only on pure cultures of organisms considered to be causing the infectious process The selection of antibiotic discs depends on the clinical consideration including the drugs that are available and in general use by the veterinarian. A tetracycline disc will predict the results against all other tetracyclines. )ulphisoxazole is a suitable representative for all the sulphonamides Aminoglycosides and quinolones should be tested separately 0encillins, )treptococci should be tested against either pencillin 6 or ampicillin. Testing against both is not necessary. 2rythromycin will predict the result of all other macrolides A clindamycin disc will predict the results for lincomycin. 7&

 .hloramphenicol, vancomycin, nitrofurantoin and trimethoprim?sulphamethoxazole are tested separately as required. A cephalothin disc will indicate the results for other first"generation cephalosporin1s 'cefalexin, cefradine, cefaloridine, cefazolin, cefapirin(. .efatozime represents ceftazidime, ceftizoxime and ceftriaxone. (.# 2nti"icro)ial sensiti0ity chart
2nti"icro)ial agent Disc content 3one dia"eter nearest -hole "" Inter"edia 9esistant S%sce,ti)le te

M- 82!T2MS 1, ^g _15 17 "1+ \1> Ampicillin 1,, ^g _15 17"1+ \1+ .arbenicillin 9 ^g _A 1,"15 \17 !ethicillin 1 ^g _1, 11"1& \15 Exacillin 1, units _&= \&A 0enicillin 1,, ^g _1> \1= 0iperacillin >9 ^g _17 \19 Ticarcillin M-82!T2MCM-82!T2M2SE IN:I*IT<9 !<M*IN2TI<NS &,?1, ^g _1A \&, Amoxycillin? .lavulanic acid 1,?1, ^g _11 1&"17 \19 Ampicillin? )ulbactam 1,,?1, ^g _1> \1= 0iperacillin? Tazobactam >9?1, ^g _17 \19 Ticarcillin? .lavulanic acid

75

!EP:EMS 5, ^g .efazolin 5, ^g .efotaxime 5, ^g .eftizoxime 5, ^g .eftriaxone 5, ^g .ephalothin $8E!<PEPTIDES 5, ^g Telcoplanin 5, ^g Mancomycin

_17 _17 _17 _15 _17 _1, _17

19"1> 19"&& 19"1A 17"&, 19"1> 11"15 19"1+

\1= \&5 \&, \&1 \1= \17 \1>

2MIN<$8E!<SIDES 5, ^g Amikacin 1, ^g 6entamicin 5, ^g Oanamycin 1, ^g )treptomycin 1, ^g Tobramycin M2!9<8IDES 19 ^g Azithromycin 19 ^g 2rythromycin TET92!E!8INES 5, ^g 3oxycycline 5, ^g Tetracycline @>IN<8<NES 9 ^g .iprofloxacin 5, ^g Balidixic acid 1, ^g Borfloxacin 9 ^g Efloxacin <T:E9S 5, ^g .hloramphenicol & ^g .lindamycin 5,, ^g Bitrofurantoin 9 ^g <ifampin &9,?5,, ^g )ulfonamides 9 ^g Trimethoprim 1.&9? Trimethoprim? sulfamethoxazole &5.>9 ^g

_17 _1& _15 _11 _1& _15 _15 _1& _17 _19 _15 _1& _1& _1& _17 _17 _1+ _1& _1, _1,

19"1+ 15"17 17"1> 1&"17 15"17 17"1> 17"&& 15"19 19"1= 1+"&, 17"1= 15"19 15"19 15"1> 19"&, 19"1+ 1>"1A 15"1+ 11"19 11"19

\1> \19 \1= \19 \19 \1= \&5 \1+ \1A \&1 \1A \1+ \1+ \1= \&1 \1> \&, \1> \1+ \1+

77

Bote <eadymade antibiotic sensitivity discs and !ueller Dinton agar are available with Di" medial Caboratory, *ndia

79

+. !<8I'<9M !<>NT IN N2TE9 S2MP8ES Introd%ction Total coliform bacteria are a collection of relatively harmless microorganisms that live in large numbers in the intestines of man and warm and cold"blooded animals. They aid in the digestion of food. A specific subgroup of this collection is the fecal coliform bacteria, the most common member being Escherichia coli. These organisms may be separated from the total coliform group by their ability to grow at elevated temperatures '7,@.( and are associated only with the fecal material of warm"blooded animals. En0iron"ental I",act The presence of fecal coliform bacteria in aquatic environments indicates that the water has been contaminated with the fecal material of man or other animals. At the time this occurred, the source water might have been contaminated by pathogens or disease producing bacteria or viruses which can also exist in fecal material. The presence of fecal contamination is an indicator that a potential health risk exists for individuals exposed to this water. 8ecal coliform bacteria may occur in ambient water as a result of the overflow of domestic sewage or nonpoint sources of human and animal waste. Escherichia coli Method of "eas%re"ent Method 1. *f the given water sample is apparently clean, the sample about 1,,^l should be spread plated directly in .oliform agar?!ac .onkey?M<: Agar in duplicates and incubated at 5>@. for 1=hrs. &. *ncase the water sample is turbid it should be serially diluted '1, 1 , 1,& , 1,5 ``1,A ( by adding 1ml of sample to Aml of sterile peptone water and after proper mixing, 1ml from the first tube should be transferred to the second tube containing Aml of sterile peptone water. )ame way serial dilution should be done up to 1,A . 5. 1,,^l of the diluted sample at tube no. 9 and + '1,9 and 1,+( should be spread plated in .oliform agar?!ac .onkey?M<: agar in duplicates and incubated at 5>@. for 1=hrs. 7. After incubation number of 0ink colonies in .oliform agar or in !ac .onkey agar?Miolet colonies in case of M<: agar should be counted and the count is calculated according to the dilution factor and expressed as cfu?ml.

7+

Inter,retation 1. 3rinking water must have , colonies ? 1,, mC &. <ecreational bathing or swimming can1t be over &,,, colonies ? 1,, ml 5. 8or domestic animal water supply fewer than &,,, colonies ? 1,, mC

/. 8a)oratory tests for f%ngal infection

To establish or confirm the diagnosis of a fungal infection, skin, hair and nail tissue is collected for microscopy and culture 'mycology(. /ltraviolet radiation 'a 4ood;s light( can help identify some fungal infections of hair because the infected hair fluoresces green S,eci"en collection )pecimens for fungal microscopy and culture may be )crapings of scale, best taken from the leading edge of the rash after the skin has been cleaned with alcohol. )kin stripped off with adhesive tape, which is then stuck on a glass slide. Dair which has been pulled out from the roots. :rushings from an area of scaly scalp. Bail clippings. )kin biopsy. !oist swab from a mucosal surface 'inside the mouth or vagina( in a special transport medium. A swab should be taken from pustules in case of secondary bacterial infection. They are transported in a sterile container or a black paper envelope

Direct "icrosco,y The material is examined by microscopy by one or more of these methods 0otassium hydroxide 'OED( preparation, stained with blue or black ink /nstained wet"mount )tained dried smear Distopathology of biopsy with special stains. !icroscopy can identify a dermatophyte by the presence of 8ungal hyphae 'branched filaments( making up a mycelium 7>

Arthrospores 'broken"off spores( Arthroconidia 'specialised external spores( )pores inside a hair 'endothrix( or outside a hair 'ectothrix(. 8ungal elements are sometimes difficult to find, especially if the tissue is highly inflamed, so a negative result does not rule out fungal infection. A yeast infection can be identified by the presence of Peast cells, which may be dividing by budding 0seudohyphae 'branched filaments similar to those of a dermatophyte( forming a pseudomycelium.

!%lt%re .ulture identifies which organism is responsible for the infection To find out the source of infection e.g. a particular animal To select the most suitable treatment. 6rowing the fungus in culture may take several weeks, incubated at &9"5,@.. The specimen is inoculated into a medium such as )abouraud;s dextrose agar containing cycloheximide and chloramphenicol. The cycloheximide is left out if a mould requires identification. 2 negati0e c%lt%re "ay arise )eca%se; The condition is not due to fungal infection. The specimen was not collected properly. Antifungal treatment had been used prior to collection of the specimen. There was a delay before the specimen reached the laboratory. The laboratory procedures were incorrect. The organism grows very slowly.

.ulture of yeasts and moulds may be due to harmless colonisation rather than infection. The infection may be secondary to an underlying skin disesase such as psoriasis. :lood tests are not useful for the diagnosis of superficial fungal infections. :ut in subcutaneous and systemic infection, several tests may be useful. .ulture Antibodies 'histoplasmosis, coccidioidomycosis( Antigen 'cryptococcosis, aspergillus, candidosis, histoplasmosis 7=

7A

1. DISE2SES 2ND M2TE9I28S T< *E !<88E!TED '9<M 8I=EST<!I 2ND P<>8T9E S.N<. 1. Na"e of the disease Actinobacillus 'Actinobacillus lignieresi.( Material to )e !ollected and Preser0ati0e if any 0us from the abscesses. 0ortion of tongue preserved in ice. )terile )wabs from the lesions. Methods of e7a"ination 1.Microsco,ical; 0us )mear made by crushing between slides, 6rams1s stain% 6ram negative cell surrounded by gram negative filamentous sulphur granules, .Stra%sA reaction; *noculation into male guinea pig produces orchitis. 5.!%lt%ral e7a"ination 8rom affected tissues and lymph nodes cultured in serum or blood agar 1. Microsco,ical; 0us granules crushed between the slides, stained by 6ram stain, 6ram 0ositive filaments arranged like mycelia surrounded by gram negative filaments. &. !%lt%ral e7a"ination .ulture of affected portion in serum or blood agar. 1. Microsco,ical; i( :lood smear stained by Ceishman1s method will reveal characteristic rod shaped organisms with truncated ends, possessing a distinct capsule and arranged singly in very short chains. ii( :lood smear stained by !ethylene :lue will evince !c 8adyean reaction . !%lt%ral; 1. .otton wool growth in broth clear supernatant &. !edusa head colonies on agar 5. *nverted fir tree growth in gelatin agar. #. *iological; 6uinea pigs and mice are used. En i?p inoculation animal dies in 7=">& hours showing characteristic lesions. :lood smear reveals characteristic organism.

&.

Actinomycosis 'Actinomyces bo is+

8resh portion of affected tissues or scrapings preserved in ice

5.

Anthrax 'Bacillus anthracis+

0eripheral blood smear 'Tip of ear ? tail( )mear from swelling )wabs from fluid exudate?blood from natural orifices ear piece or muzzle in two or three pieces :lood stained soil

9,

7.

:lack disease 'Clostridium no yi+

0ieces of liver in sterile container

9.

:lack quarter ':.a( 'Clostrldium chau oei+ and 'Clostridium septicum+

)mears from the swelling !uscle impression smear. !uscle piece"air dried

+.

:otryomycosis 'Staphylococcus aureus+

0us smear

1. 3emonstration of anaerobic bacteria with sub"terminally located oval spores by gram staining . Anaerobic culture in <obertson cooked meat medium and identification 1. Microsco,ical; !uscle impression smear stained by .laudius method of staining will reveal rods with sub"terminal spores. . *iological; *ntramuscular in#ection into a guinea pig produces characteristic gangrenous necrosis and death. 2xtensive hemorrhagic edema and emphysema with s?c haemorrhage is seen. *n#ection of a drop of 9F calcium chloride before inoculation accelerates the reaction. Clostridium chau oei and clostridium septicum can be differentiated by cultural characters and biological test by toxin neutralization test. 1. 3emonstration of gram positive .occi in clusters ..ulture and identification on !annitol salt agar, !altose Agar and :lood agar I :eta hemolytic, Dot .old 0henomenon #..oagulation of plasma, Bovobiosin sensitivity are the characteristic features of pathogenic organism. 1. Microsco,ical; )mears stained by 6ram1s staining reveal 6ram negative coccobacilli. 3ark pink stained cocobacilli in blue backround with !odified Viehl Beelsan staining. . !%lt%ral; :rucella selective agar, 0otato 3extrose agar, Tryptose agar, )erum dextrose agar or liver infusion agar can be used. 9 "1,F increased tension of carbon dioxide favours the growth of B. abortus. #. Stra%s test. ; *ntraperitoneal inoculation into a male guinea pig produces orchitis I 7. Serological 'a( <apid plate test I for screening 'b( Tube agglutination test I for confirmation 'c( A:< test or !<T test I

0us swab

>.

:rucellosis 'Brucella abortus" occurs commonly in cattle B. melitensis occurs commonly in goats and B. suis I occurs commonly in pigs.(

)erum preserved in 9F chloroform !ilk form affectd quarter )mears form vaginal exudates and foetal stomach contents )wabs from vaginal exudates or foetal stomach contents, foetal cotyledons and uterine discharge

91

=.

:ovine Cymphangitis '.ersinia pseudotuberculosis type 111+ .aseous Cymphadenitis, .aseous Cymphangitis 'Corynebacterium pseudotuberculosis+

to screen milk form affected animals. 0us smear?smear from 1.Microsco,ical 0us smear on 6ram1s staining reveals 6ram negative rods. gland .*solation and identification of the 0us swab organism in JC3or :rilliant 6reen Agar. #.*iological; )traus test )wabs from abscesses. 1.Microsco,ical )mear from caseous lesions on 6ram1s staining reveals 6ram positive pleomorphic bacterium with metachromatic granules demonstrated by 2l)erts staining . *solation and identification of the organism from :lood agar. 3ifferentiation based on non growth of bacteria on !ac .onkey agar. !ere isolation and identification of this nonsporulating 6ram negative, anaerobe do not confirm the etiological aspect. :iological test in rabbits is always necessary as this organism appears as secondary invader in most of the cases. !%lt%ral; *solation and identification of the organism using selective media and characterization. 1. !icroscopical 3emonstration of bipolar organism by giemsa?leishman staining. . *solation and identification of the organism in :lood agar ' tiny colonies( and non growth of bacteria on !ac .onkey agar and confirmation #. :iological test in mice and rabbits. 1. .ulture and identification of the organism in 00CE medium supplemented with &, F Dorse serum, penicillin and Thalous acetate. . 0late agglutination test to demonstrate the antibodies from serum of ailing animals

A.

1,

.alf 3iphtheria '-usobacterium necrophorum+.

)wab from the lesions.

11.

1&.

.alf 3ysentry or white scour or .olibaciliosis. 'Escherichia coli+ .alf 0neumonia 'Pasteurella multocida+ )heep pneumonia 'Pasteurella haemiolytica+ .ontagious :ovine pleuro pneumonia '.:00( 'M.mycoides subspecies mycoides 'small colony types(

<ectal swabs

)wabs from pleura and lungs.

15

0iece of lung in ice and )erum 0iece of lung in ice

.ontagious .aprine

9&

17.

pleuro pneumonia '..00( 'M.capricolum subspecies Capri pneumonia '85=(, .hronic respiratory disease 'Mycoplasma gallisepticum+

Tracheal impression smear, Trachea in ice, )wabs from nasal secretion

)erum from the infected birds 19. 2nterotoxaemia. 'Clostridium 0elchii 5ype 4,( *ntestinal contents or a loop of intestine tied at both ends and preserved in ,.9F .hloroform.

1. 3emonstration of pleomorphic fungi like organism from the impression smears by 6iemsa staining techniques .8or culture and identification of the organism in 00CE medium supplemented with &, F Dorse serum, penicillin and Thalous acetate #.Plate aggl%tination test ; To demonstrate the antibodies from serum of ailing birds 1. *iological test I in mice by caudal vein inoculation chloroform treated intestinal content after centrifugation . 3eath of mice with neurological symptoms in few minutes to two hours. . To7in ne%tralization test Actual toxin can be determined by neutralization with specific antitoxins. 1. !allein test in living animals. .)traus test positive. Microsco,ical ;The organisms can be demonstrated by 6iemsa strain or silver impregnation staining techniques and could be viewed under 38! 1.Microsco,ical ;:ipolar organisms could be detected by 6iemsa staining

1+. 1>.

6landers 'Bur,holderia mallei+ 8owl spirochaetosis 'Borrelia anserine+

)mears from discharge :lood buffycoat smears 0iece of liver and spleen ice :lood smear :lood swab in charcoal medium Cong bone packed in table salt

1=.

8owl cholera '0.multocida(

1A.

Daemorrhagic septicemia. 'D.).( 'Pasteurella multocida+

..ulture and identification in :lood agar and !ac .onkey ' Bo growth of :acteria( #. *iological test ; :one marrow from long bone will be in#ected in to mice. 3eath of mice occus in &7 to 7=hrs Auricular vein smear. Microsco,ial; )mears stained by Ceishman1s stain reveal bipolar Eedema fluid organisms. smear. .!%lt%ral; *solation and identification Deart blood smear Deart blood swabs. identification in :lood agar and !ac )wabs from internal .onkey 'Bo growth f bacteria5 95

&,.

Nohne1s disease '0aratuberculosis( 'Mycobacterium a ium sub sp Paratuberculosis+

organs Cong bone preserved in charcoal in case of extensive putrification. <ectal pinch smear and <ectal washings.

#. *iological Test; *n mice and rabbits.Daemorrhagic tracheitis in rabbits.

1. Microsco,ial; )mear stained by Viehl Beelsan1s method reveals acid fast bacteria. . .ultural examination in 3orset1s 2gg or C.N. medium. #. *iological test Nohnin or Avian tuberculin test in living animal. 1. 3emonstration of motile spiral organism by 3ark field !icroscope '38!( and in silver impregnated staining technique. .!icroscopic Agglutination Test '!AT( in microtitre plates.

&1.

Ceptospirosis 'Leptospira interohaemorrhagia e and L. canicola occur in dogs and produce #aundice. L.. pomona has been incriminated in bovines.( Cisteriosis 'Listeria monocytogenes+.

)erum% /rine.

&&.

:rain, nasal swab and heart blood swab Deart blood of the foetus preserved over ice.

1. *solation and identification of the organisms in Cisteria selective agar, :lood agar incubated at &,@. . Microsco,ical ;. )mall pleomorphic 6ram positive rod, motile, beta haemolytic and .A!0 positive. ' *solation is augmented by prior chilling in ice(. 1. Microsco,ical ;. 3emonstration of 6ram positive cocci in chais and clusters and some pleomorphic organisms. . *solation and identification of the organisms by growing it in 2dwards medium, mannitol salt Agar and blood agar. 1. *solation and identification of the organism in JC3, :rilliant 6reen agar and :ismuth sulphite agar. . Agglutination test with )almonella 0ullorum .oloured Antigen ')0.A(

&5.

!astitis 'Str. agalactiae, Str. dysagalactiae/ Str. uberis/ Staph. aureus/ Cory. Pyogenes+ 0ullorum disease S fowl typhoid. 'Salmonella pullorum and Salmonella gallinarum+

)mears from milk sediment. !ilk from suspected cases of mastitis

&7.

.loacal swab. Ailing chicks. )erum from birds. 0iece of intestine tied at both ends intestinal swab. 97

&9.

)almonellosis

&+.

)trangles 'Streptococcus e3ui+

3epending upon the disease conditions swab should be collected. )wabs from abscess.

1.*solation and identification of the organism in JC3, :rilliant 6reen agar and :ismuth sulphite agar. 1. Microsco,ic ; 3emonstration of chains of gram positive cocci in grams staining. . The isolation and identification of Streptococcus e3ui in Ed0ards medum and in blood agar. .A!0 positive 1.*solation and identification of the organism " Bon hemolytic pin point colonies on blood agar. ..oagulase positive organism grow as bottle brush like pattern in gelatin agar. 1. Mor,hology ;C.tetani organism '3rumstick appearance with terminal oval bulged spores( . *iological ;To demonstrate neurotoxin in mice #. *solation and identification of the organism in :lood agar and in <oberson cooked meat medium. 1.Microsco,ical e7a"ination.; 3emonstration of Acid fast organism by Viehl neelsan staining. .!%lt%ral e7a"ination; .ulture develops in 7 to = weeks in 3orset egg medium or Cowenstein Nensen medium. #.*iological test; *n guinea pigs, rabbits and fowl depending upon the type. 3isease is reproduced in 7 to = weeks. 7. T%)erc%lin test ;.*ntradermal test in live animals '3evelopment of inflammatory reaction at in#ection site( 3emonstration of .hlamydial elementary bodies by 8A, 6iemsa and !VB stains. *solation in Polk sac of fertile eggs.

&>.

)wine 2rysipelas 'Erysipelothri" rhusiopathiae+ Tetanus 'Clostridium tetani+

Affected organs )erum

&=.

)mear from lesions )erum from the affected animal Becrotic wound tissues

&A.

Tuberculosis 'Mycobacterium bo is# Mycobacterium tuberculosis# Mycobacterium a ium+.

)mear from lesions. )wabs from lesions. Cesion preserved in ice.

5,.

0sittacosis 'Chlamydia psittaci+ a!!ecting psittacine and domestic birds

*mpression smears from the organs .on#unctival swab

99

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=eterinary Micro)iology and Micro)ial disease " 0.N. auinn, :.O. !arkey, !.2. .ater, 4.N. 3onnelly and 8... Ceonard, :lackwell )ciences Ctd., 2d. &,,& Identification of *acterial S,ecies " Oimberley .hristopher and 2lsa :runo/ 3epartment of :iological )ciences, /niversity of Alberta, 2dmonton, Alberta, .ABA3A NN':S F8a)oratory Man%al and Proced%res 4 'ro" internet5 I '.hapter"9( I Nason woodland, 0inetop, Arizona 2ni"al Micro)iology Edn.411+/5 I:uxton and 8rasier Ne) ; Introd%ction to )acteria O )cience in the real world !icrobes in action '1AAA( !linical =eterinary Micro)iology 411145 " 0.N. auinn, !.2. .arter, :.O. !arkey and 6.<. .arter Internet So%rce ;http ??users.stlcc.edu?kkiser?biochem.html Internet So%rce ; http ??www.microbelibrary.org?A)!Enly?details.asp Internet So%rce " http ??www.microbiologyprocedure.com

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