bacte lab final practical

8/14/2019 Bacte Lab Final Practical

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HEKTEON ENTERIC AGAR (HEA)

This is both a selective and differential culture medium which is used to isolate and

to differentiate the lactose fermenting from non-lactose fermenting Gram (-) bacilli.

Inhibitors bile salts and citrate

pH indicator bromthymol blueHydrogen sulfide indicator ferric ammonium citrate with a thiosulfate

!arbohydrates lactose" sucrose" salicin

#lue to green colonies without blac$ center of non-lactose fermenting and non-

hydrogen sulfide producing Gram(-) enteric bacilli li$e%

Providencia rettgeri

Morganella morganii

Shigella dysenteriae

#lue to greencolonies with black centerof non-lactose fermenting but hydrogen

sulfide producing Gram(-) enteric bacilli li$e%

Salmonella type

Proteus vulgaris

Proteus mirabilis

&in$ to redcolonies with black centerof late lactose fermenting Salmonella li$e%

Salmonella arizonae

'ellow to colorless colonies with black center of non-lactose fermenting

Salmonella li$e%

Salmonella typhiSalmonella typhimurium


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XYLOSE LYSINE DEOXYCHOLATE AGAR (XLD)

This is a selective and differential medium which is used to isolate and differentiate

the Salmonellaand Shigellaspecies from other coliforms.

Inhibitor deoycholate

pH indicator phenol red

Hydrogen sulfide indicator ferric ammonium citrate with sodium thiosulfate

!arbohydrate ylose

mino acid - lysine

'ellow colonies with black center of ylose fermenting and hydrogen sulfide

producing Gram(-) enteric bacilli li$e%

Citrobacter freundii

Proteus mirabilis

Proteus vulgaris

*'ellow colonies without blac$ center of ylose fermenting but non-hydrogen sulfide

producing Gram(-) bacilli li$e%

Escherichia coli

Serratia marcescens

Klebsiella pneumoniae

Enterobacter aerogenes

Providencia rettgeri+ed colonieswithout blac$ center of non-ylose fermenting Gram (-) enteric bacilli

li$e%

Shigella dysenteriae

*+edcolonies with black centerof ylose fermenting" hydrogen sulfide producing

but lysine decarboylase positive Gram(-) enteric bacilli li$e%

Salmonella typhi


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SALONELLA SHIGELLA AGAR (SSA)

This is a highly selective and differential medium which is used to isolate and

differentiate the species of Salmonellaand Shigella.

Inhibitors brilliant green" bile salts" citrate

pH Indicator neutral red

Hydrogen sulfide indicator ferric ammonium citrate with sodium thiosulfate

!arbohydrate lactose

'ellow to orange colonies with black centerof lactose fermenting and hydrogen

sulfide producing Gram(-) enteric bacilli li$e%


Salmonella arizonae

*'ellow to orange colonies without blac$ center of lactose fermenting but non-

hydrogen sulfide producing Gram(-) enteric bacilli li$e%

Escherichia coli

Klebsiella pneumoniae


Shigelle sonnei

Serratia marcescens

&in$to red colonies without blac$ center of late lactose fermenting Shigellali$e%

Shigella sonnei

'ellow to colorless colonies without blac$ center of non lactose fermenting Shigella

li$e%

Shigelle dysenteriae

Shigelle flexneri

Shigelle boydii


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!IS"TH S"L#ITE AGAR (!SA)

This is a highly selective medium for the isolation of Salmonellatyphi.

Inhibitors bismuth sulfite and brilliant green

!arbohydrate glucose!lack with metallic sheen colonies of Salmonella typhi

,ar$ green colonies of uninhibited Gram(-) enteric bacilli

THIOS"L#ATE CITRATE !ILE SALTS S"CROSE AGAR (TC!S)

This is a highly selective medium which is used to isolate and to differentiate the

sucrose fermenting from non-sucrose fermenting species of Vibrio.

Inhibitors thiosulfate" citrate" bile salts" and high pH

pH indicator bromthymol blue

!arbohydrate - sucrose

'ellow colonies of sucrose fermenting Vibrio li$e Vibrio cholerae and Vibrio

alginolyticus

#luegreencolonies of non-sucrose fermenting Vibrioli$e Vibrio parahaemolyticus


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ANNITOL SALT AGAR (SA)


to differentiate the mannitol fermenting from non-mannitol fermenting species of

Staphylococcus

Inhibitor ./ a!l


!arbohydrate mannitol

'ellow colonies of mannitol fermenting Staphylococcusli$e%

Staphyloccocus aureus

&in$ colonies of non-mannitol fermenting Staphylococcus li$e%

Staphylococcus epidermidis

Staphylococcus lugdunensis

Staphylococcus saprophyticus

+ed pigmented colonies of Serratia marcescenson 0ac!on$ey agar


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***1warming colonies of Proteus vulgaris***

***#lue-green pigmented colonies of Pseudomonas aeruginosa on nutrient agar

plate

(,iffusable pigments blue pyocyanin2 green pyoverdin)***

&in$ to purple with greenish metallic sheen colonies of lactose fermenting

Escherichia coli


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3arge pin$ mucoid colonies of lactose fermenting Klebsiella penumoniae

EOSIN ETHYLENE !L"EAGAR (E!)


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differentiate the lactose fermenting from non-lactose fermenting Gram(-) bacilli.

Inhibitors eosin and methylene blue

pH indicators eosin and methylene blue

!arbohydrate - lactose

&in$ to purple colonies with $ark center(fish eye colonies) of lactose fermenting


!olorless non-lactose fermenting colonies on 40# plate li$e those of Salmonella

typhi Shigella dysenteriae Providencia species"Morganella species"etc.


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ACCONKEY AGAR


to differentiate the lactose fermenting from non-lactose fermenting Gram (-) bacilli.

Inhibitors crystal violetand bile salts

pH indicator neutral red

!arbohydrate - lactose

&in$to red colonies of lactose fermenting Gram (-) enteric bacilli

'ellowto colorless colonies of non-lactose fermenting Gram (-) enteric bacilli


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"REA !ROTH

This li5uid medium is used to identify Gram (-) enteric bacilli based on their ability

to produce the en6yme urease withing 7 to 8 hours (rapid) or within 78 hours (late)

of incubation.


1ubstrate urea

If the organism produces urease" this splits urea into ammonia" carbon dioide" and

water. mmonia is then converted into an al$aline compound ammonium carbonate

which changes the color of the medium to pin$-red.

+apid urease producers (positive within 7 to 8 hours of incubation)%

Proteus vulgaris

Proteus mirabilis

Morganella morganii


3ate urease producers (positive within 78 hours of incubation)%


Klebsiella pneumonia

!ersinia enterocolitica

Serratia marcescens.


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ETHYL RED% &OG"ES 'ROSKA"ER (R%&') !ROTH

This li5uid medium is also $nown as peptone glucose broth and is used to identify

the Gram (-) enteric bacilli based on the ability of the organisms to ferment glucoseto pyruvic acid by one of two pathways" mied acid fermentation pathway which is

tested by the 04TH'3 +4,T41T and the #utylene Glycol &atch which is tested by

the 9:G;41 &+:1


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pH indicator bromthymol blue

1ource of carbon sodium citrate

o carbohydrate or protein component

LYSINE IRON AGAR (LIA)

This tubed medium is used to identify the Gram (-) enteric bacilli based on the

following biochemical characteristics%

3ysine deamination indicated by red slant

3ysine decarboylation and glucose fermentation indicated by purple butt

Glucose fermentation only indicated by yellow butt

Hydrogen sulfide production indicated by blackeninof the medium

pH indicator bromcresol purple

Hydrogen sulfide indicator ferric ammonium citrate with sodium

thiosulfate

!arbohydrate glucose

mino acid lysine

&eptone


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TRI'LE S"GAR IRON AGAR (TSI)

This tubed medium is used to identify the Gram (-) enteric bacilli based on the

following biochemical characteristics%

Glucose fermentation indicated by yellow butt

3actose fermentation indicated by yellow slantHydrogen sulfide production indicated by blackeninof the medium

Gas production indicated by presence of a crac$" bubble or gas space


Hydrogen sulfide indicator ferric ammonium citrate with sodium

thiosulfate

!arbohydrate ?/ glucose" ?=/ lactose" ?=/ sucrose

&eptone


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!ACTERIOLOGIC ANALYSIS O# ATER

0ost &robable umber 0ethod (0&) or 0ultiple Tube Techni5ue

&resumptive Test

set of three tubes of #rilliant Green #ile 3actose #roth(#G#3#) and water sample

Tube no. ? with ?= ml (double strength) #G#3# and ?=m3 of water sample

Tube no. 7 with ?= ml (single strength) #G#3# and ?.=m3 of water sample

Tube no. > with ?= m3 (single strength) #G#3# and =.?m3 of water sample

(small tube is called ,urham@s fermentation tube)

TY'ICAL REACTIONS ON S"L#IDE INDOLE OTILITY EDI" (SI)

?. Hydrogen sulfide positive

Indole (A)

0otile

&ossible organism% Proteus vulgaris

7. Hydrogen sulfide (-)

Indole positive0otile

&ossible organisms%

Citrobacter "oseri

Escherichia coli



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Morganella morganii

>. Hydrogen sulfide (-)

Indole (-)

on motile

&ossible organisms%

Klebsiella pneumonia

Shigella sonnei

S"L#IDE INDOLE OTILITY EDI" (SI)

This semisolid medium is used to identify Gram (-) enteric bacilli based on the

following characteristics%

Hydrogen sulfide production indicated by blackeninof the medium

Indole production indicated by red color of the medium

0otility indicated by diffusion of growth away from the point of inoculation


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COAG"LASE TEST SET%"'

!oagulase test differentiates the pathogenic Staphylococcus aureusfrom the non-

pathogenic Staphylococcus epidermidis and Staphylococcus saprophyticus

1lide test plasma plus specimen

&ositive (A) visible clumping

egative (-) no visible clumping

Tube test plasma plus specimen

&ositive (A) visible clot or coagulum

egative (-) no clot or coagulum

&athogenic Staphylococcus aureus &ositive (A)

on-pathogenic Staphyloccocus epidermidis and Staphylococcus

saprophyticus egative (-)

CHOCOLATE AGAR

n enriched medium that can be used to isolate organisms re5uiring comple

nutrients such as #aemophilus


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4nriching substance is heated blood.

!LOOD AGAR 'LATE (!A')

This is an enriched medium that can be used to isolate both Gram positive and

Gram (-) cocci and bacilli. The enriching substance is preferable unheated /

defibrinated sheep blood.

This is also classified as differential culture medium because it differentiates

organisms based on their hemolytic patterns ehibited on this medium.

lpha hemolytic if colony is surrounded by an incomplete 6one of hemolysis

which is translucent and green in color

#eta hemolytic if colony is surrounded by a complete 6one of hemolysis

which is clear and transparent

Gamma hemolytic if colony is not surrounded by any 6one of hemolysis2

there is no change in the surrounding medium.

CATALASE TEST SET%"'!atalase test uses >/ hydrogen peroide. The positive

reaction is indicated by bubbling or effervescence.

&ositive (A) reaction is given by%

ll Staphylococcusspecies

ll Gram (-) enteric bacilli ecept Shigella

dysenteriae


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egative (-) reaction is given by%

ll Streptococcusspecies

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