prac revision 2011 EPHM download

8/3/2019 prac revision 2011 EPHM download

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Practical Revision EPHM tute

EPHM 2011

Biochem tests

Citrate test

What do you look at first?

An "assimilation test means you look for .......

Positive tests show........ and ...........

Test useful in ID to split.......... and ...............(A) may be ................. (B) may be ...............

A B

Indole test

The amino acid is ..................

The reagent is .......................

Useful to split ........... and .....................

Also splits .................. and ..................Also splits .................. and ..................



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Biochem tests

Motility

The red compound is insoluble – why is thisimportant?

(A) is pos/neg(?) and may be ...................

(B) is pos/neg(?) and may be ..................

A B

A B

ONPG test

Yellow compound is cleaved by the sameenzyme which cleaves .....................

Why then can a NLF be ONPGPOS?

(A) is pos/neg and could be .............(B) is pos/neg and could be .............

Biochem

A

B A B

Phenylalanine deaminase

Useful for ID of .............. which is pos/neg

Reagent added is ....................

(A) is pos/neg (B) is pos/neg

pos/neg colours similar, therefore test ....&....

Urease test

Useful for ID of .......... which is strong pos.

Splits the NLF pathogen ................. .(neg)from ..................................................(pos)



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Biochem

Test is ...........................................................

Base media purpose is ...............................

ODC colour spot is ...... and test here is .......

ADH colour spot is ...... and test here is .......

LDC colour spot is ...... and test here is .......

Base result is satisfactory/unsatisfactory

ADH result is ..............................

LDC result is .............................

ODC result is ............................

Biochem tests (no pics)

Methyl red test

Voges Proskaur

Gelatin liquefaction

Nitrate reduction test

(Gram stain

Catalase

Oxidase)



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SIM

Motility = ....................

H2S = ........ Indole = ........

Based on H2S and motility,it may be .................

Motility = ....................

H2S = ........ Indole = ........

Based on H2S, indoleand motility, it may be...................................

Motility = ....................

H2S = ........ Indole = ........

Based on motili ty,it may be .................

O/F test (Hugh & Leifson's)

Which is Escherichia coli, which is Pseudomonas aeruginosa ?



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KIA

Which is Escherichia coli, which is Pseudomonas aeruginosa ?

KIA

Pseudomonas aeruginosa Unknown GNR, Catalase pos, oxidase neg.

Explain the discordant glucose results. Possible ID = ...............................

What is the purpose of the FeSO4.7H2O in KIA?



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KIA – early and late stages – no. 1

Salmonella typhimurium

test

nb. Slight yellowing of KIA slant early

Why does this happen?

What happens later?

EarlyLate

KIA – early and late stages – no. 2

Proteus sp. (NLF)



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API20E

Identify which is Proteus, Salmonella and Citrobacter.



8

Laboratory Investigation of food-borne disease – (some components only!)

• Food ! maceration & resuscitation in BPW (resuscitation = ...................................?)

• Food suspension ! possibly plate directly to selective and differential agars (“food agars”) – to grow pathogen and suppress non-pathogens; or differentiate these

– PEMBA; Baird-Parker; MSA; XLD; SSA; BSA; MAC; TCBS; TSC; Oxford; (poss. HBA)

– (QC all these agars with appropriate reference strains – to ensure agars working properly)

• Food suspension ! enrichment (grow pathogen; suppress other orgs)

– alkaline peptone water (APW) enrich Vibrio spp. and Aeromonas spp.

– mannitol cysteine selenite broth (MCSB) enrich Salmonella spp. and most Shigella spp.

– (? cold enrich for Listeria monocytogenes )

• Plate enrichment broths! selective and differential agars for growth

– alkaline peptone water (APW)! TCBS (HBA)

– mannitol cysteine selenite broth (MCSB)! XLD; BSA; MAC; SSA (HBA)

• Read plates and count/follow-up suspicious colonies:

– Rapid tests – Gram, catalase and oxidase

– Latex coagulase on suspicious staphylococci – s/c suspicious colonies to HBA back-up cultures (with O129 disk for poss. Vibrio spp.)

• Identification and confirmatory testing:

– Biochemical ID (API strips or biochem bottles); lipid/spore stain for .........; tube coag for .........

– O (LPS) and H (flagella) serogrouping for Salmonella isolates

• Refer isolates to reference lab:

– for strain typing – to show if outbreak strains are related or not & for epidemiological tracking

Epidemiological investigation in outbreaks (some points only!):

• Coordinationbetween:

– Food lab – isolates bacterial and viral pathogens from food

– Clinical lab – isolates pathogens from patients

– Public Health Unit (PHU) – investigates, traces patients and coordinates

– NSW Food Authority – inspects and prosecutes

• Inspection of suspect premises

– swabs and sampling for Food Lab investigation

• Questionnaire

– for cases and controls involved in the outbreak

• Questionnaire data: – Epidemic Curve

• Point source vs intermittent/recurrent transmission

• Food intoxication vs food infection

– Food Attack Table

• for statistics

– Chi-squared test

• “association” or “dependency” between factor and outcome (or lack thereof)

• (or, is there an over-association between the two beyond that predicted by random chance)

– Odds Ratio and CI

• magnitude of risk for outcome attributable to a factor, compared to its absence

• (or, how much does eating the food increase disease risk, compared to not eating it?)



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Outbreak 1

What are important plate features? What to do now?

Outbreak 2 isolates on Baird Parker Agar

Points – plate appearances (and reasons why), which is suspicious,and what happens next.

0-1-)#%.&2(3%&1#'(.%&-,&1%.%*2()(.%&2(1-&.4,,-4)+%+&56&',-*%1670&01%(,#)89&:1(0$&+4%&*-&*%14,#*%&',-+407-)9&



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Outbreak 2 – cook's nose

HBA(which arestaph. col?)

whichMSA?

whatnext?

Outbreak 2 – Staph. isolates from cook and assistant on MAC

!"#$%&'(#)*&

+,-'./

<=&.(1*&>&3%,6&

2#82&.(1*

;4.'#0#-4.&

3*''1+"4$'"'56(&3('+(&

@AB$/0(4(00$1'$#10:'-/10,1'-$02$*/&(/07$C$02()1#(&31#102

.*'$#"$.*+*4*0($+(0+7$02*/&(/0$5$89*0($)"01:;($5$%&(*<0$#"='$:00/(2$



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Outbreak 3 – Clostridium perfringens

Outbreak 4 – Salmonella

MAC (anything suspicious?) SSA

*'*(&"%1.*44E$1'./%*+(#2$"&-*'103$10$,(3"4E:.2$

!""#$%"&'($)*+,"-('

#"/%4($D"'($"6$,(3"4E010$

.*/0(0$-*'-(&1'(2$

4*&-($9F!$$$

)1'<$."4"'1(0$'"+$0/0)1.1"/0$



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Outbreak 4 – BSA

BSA: Citrobacter and Salmonella typhimurium Criteria for suspicious colonies ..............................................

Outbreak 4 - XLD

Proteus and E. coli isolatesAnything suspicious?Reasons for plate appearance.........................

.1+&"%*.+(&

(&E$03*44$GHI

GHI$0+*1'1'-$+,($*-*&2$

>#(01-'(#$6"&$0*43"'(44*?$

Reasonforyellow-sucrose&/orlactose



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Outbreak 4 - XLD

XLD (fresh ", and old!)Reasons for plate appearance ...........................

Suspicious? What next?

Outbreak 5 – Vibrio parahaemolyticus

Rectal swab – TCBS

What's suspicious & what next?

Rectalon MAC

Rectalon SSA

BlackH2S-mightbeLDCposi>ve(?)lactose(-)sucrose(-).

lactoseneg-pinkalkalinecolonies.Ifaskedwhythecolonieshavetheappearanceontheagar.

Doacatalse,oxidasetest.API(organismissalmonella)

Bigonesare



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Outbreak 5

Oyster APW enrichmentyielded this isolate on TCBS

E. coli, Citrobacter and Enterococcus on TCBS

Outbreak 5

Vibriostatic disk contains ........................API20NE strip showing ...................... tests

ucrose(+)

Sucrose(-)

Suspiciouscolonies

Assimila>ontest-whethertheorg.cangrowoncarbohydratealone.



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Pathogenic Vibrio spp.

V. cholerae V. parahaemolyticus

Listeria monocytogenes

Oxford media HBA



16

Disinfectant testing

Points –Category A test: kill over 8 minutes for 2 inoculums added at 0 & 10 min.“Hospital Grade” disinfectant

Category C test: kill over 8 min. for 1 inoculum

Organisms: standard reference strains of relevant pathogens

Inoculum: 2 x 108 – 2 x 109 cfu/ml

Pass criteria: – growth in no more than 3 of 5 recovery tubes inoc.with 0.02ml each

– passes as above on repeat testing over 3 consecutive days

Controls: Sterility control (recovery broth)Sterility control (disinfectant)Viability of organisms controlDisinfectant inactivator control

Initial pre-clean increases disinfectant efficacy as biological material inhibits disinfectants

Water & Food Faecal coliform Quality Testing

Rationale of “indicator organisms” – what are they and how chosen?

Plate counts for total heterotroph numbers

R2A agar: better for enviro oligotrophs and injured/damaged bacteria. Low nutrientTSA and PCA plates: for standard heterotrophs

Membrane filtration of water for total orgs., and total & faecal coliformsTotal count: if high indicative of high [nutrient]? – if so why; & what orgs present

volumes handledusing M-Endo plates for presumptive coliforms and faecal coliforms on membranesconfirmatory EMB + indole for faecal coliforms

MPN for water and foods (for faecal Escherichia coli )

LT (lauryl (SDS) tryptone/gas) as selective and presumptive for coliformsEC as more selective for faecal E. coli (inhibitory formulation; temp. 44#C)Confirmation via EMB appearance and indole

Consider permissible numbers of faecal coliforms, concept of "indicators", use of faecalEnterococci , and time req'd for testing

INEXAMMMMMMMMMMMMMMMMMMMMMMMMMM

BEAWAREOFWATERQUALITYMETHODS



Lastly,

Throat isolate, HBA rectal isolate, BHIA

prac revision 2011 EPHM download

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