M Activity 3 - Identification of gram negative bacilli PDF

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Identification of gram negative bacilli...

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MLS 4455 / 5555 Student Laboratory Practices Lab Activity #2 – Identification of Gram Negative Bacilli (growing on MacConkey) / Urine Culture Workups / Stool Culture workups Due March 16th

Introduction: In this lab we will perform, observe and interpret various identification tests for GNB identification. We will interpret TSI and LIA tubes as well as read urine and stool cultures. Objectives: Upon completion of this lab and suggested reading materials a student should be able to: 1. 2. 3. 4.

Recognize lactose and non-lactose fermenters on MacConkey agar. Learn the characteristics defining the family Enterobacteriaceae. Perform and interpret basic identification tests for various Gram negative bacilli. Interpret Triple Sugar Iron (TSI) and Lysine Iron Agar (LIA) tubes which may be used for screening of potential stool pathogens. 5. Learn to perform the motility test using a coverslipped drop. 6. Identify contaminants and pathogens in the urine cultures. 7. Identify normal flora and pathogens in the stool cultures.

Section one.Identification of GNR(growing on MacConkey) Process:  Perform oxidase test  Describe colony appearance on MacConkey agar: lactose or non-lactose fermenter (LF and NLF)  Observe positive and negative motility and record your findings  Become familiar with the various biochemical tests used to identify routine gram negative bacilli. MicroScan identification panels will be used.  Fill out reactions for select Gram Negative Bacilli. Activity: Oxidase test:

Reaction

E. coli

Pseudomonas aeruginosa

Proteus mirabilis

Negative

Positive

Negative

Motility by coverslipped drop method

Reaction

E. coli

Klebsiella pneumoniae

Motile

Non-motile

Screening gram negative bacilli using TSI and LIA slants (IF AVAILABLE at your site) E coli

Salmonella

Proteus mirabilis

TSI

LIA

TSI

LIA

TSI

LIA

A/A, no H2S gas produced

K/K, no H2S gas produced

K/A, H2S gas produced

K/K, H2S gas produced

K/A, no H2S gas produced

R/A, no H2S gas produced

Gram negative bacilli identification reactions by MicroScan Panels. 1. Organism: Pseudomonas aeruginosa Appearance on MacConkey:___Non-lactose fermenting colorless colonies __________ Oxidase reaction: __Positive ________ MicroScan identification panel: GLU -

RAF -

INO -

URE -

LYS -

TDA -

CIT +

TAR +

OF/G +

SUC -

RHA -

ADO -

H2S -

ARG +

ESC -

MAL +

ACE +

NIT +

SOR -

ARA -

MEL -

IND -

ORN -

VP -

ONPG -

CET No growth

DCB

2. Organism:E.coli PAGE 1

Appearance on MacConkey:___Lactose fermenting pink colonies_______ Oxidase reaction: __Negative _______ MicroScan identification panel: not provided GLU

RAF

INO

URE

LYS

TDA

CIT

TAR

OF/G

SUC

RHA

ADO

H2S

ARG

ESC

MAL

ACE

NIT

SOR

ARA

MEL

IND

ORN

VP

ONPG

CET

DCB

3. Organism: Klebsiella pneumoniae Appearance on MacConkey:__Lactose fermenting pink diffused mucoid colonies_________ Oxidase reaction:___Negative__________ MicroScan identification panel: GLU +

RAF +

INO +

URE +

LYS +

TDA -

CIT +

TAR +

OF/G +

SUC +

RHA +

ADO +

H2S -

ARG -

ESC +

MAL +

ACE -

NIT +

SOR +

ARA +

MEL +

IND -

ORN -

VP +

ONPG +

CET No growth

DCB

4. Organism: Proteus mirabilis Appearance on MacConkey:__non lactose fermenting round colonies_________ Oxidase reaction: _Negative___________ MicroScan identification panel: Not provided GLU

RAF

INO

URE

LYS

TDA

CIT

TAR

OF/G

SUC

RHA

ADO

H2S

ARG

ESC

MAL

ACE

NIT

SOR

ARA

MEL

IND

ORN

VP

ONPG

CET

DCB

5. Organism: Citrobacter freundii(if available at your site): Not available PAGE 2

Appearance on MacConkey: __________ Oxidase reaction: ____________ MicroScan identification panel: GLU

RAF

INO

URE

LYS

TDA

CIT

TAR

OF/G

SUC

RHA

ADO

H2S

ARG

ESC

MAL

ACE

NIT

SOR

ARA

MEL

IND

ORN

VP

ONPG

CET

DCB

Questions: 1. The VP test detects which end product of glucose fermentation? 2,3 –Butanediol and Acetoin

2. List 2 organisms that can be used to quality control (QC) the indol test? Positive control – E.coli Negative control - Pseudomonas

3. Which test on the MicroScan panel best distinguishes an enteric gram negative bacillus (Enterobacteriaceae) from a non-enteric gram negative bacilli (non-Enterobacteriaceae)? Glucose fermentation test can distinguish enteric gram negative bacilli

4. What is a frequent characteristic of Proteus species observed on blood agar? Swarming growth with yeast like smell

Section Two.Urine Culture Workup

PAGE 3

Process:  Examine culture plates  Perform necessary tests to confirm or rule out a pathogen  Read MicroScan panel if necessary  Make your conclusion Activity: Case 1:56 yr o female present to ED with low abdominal pain. Urine culture Clean catch Mixed or pure culture?

Pure

Predominant?

N/A

Colony count for predominant CFU/ml

>100000 CFU/ml

Growth: BAP yes MAC yes BEA (if available)

Small round yellow orange colonies Small round pink colonies (lactose fermenter)

Rule out potential pathogen – Non lactose fermenting organisms and Gram positive organisms GS result

Gram negative rod

Oxidase

Negative

Final report: E. coli isolated with >100000 CFU/ml after incubation of 48 hrs

Appearance on MacConkey: __Pink (Lactose fermenter)_small round colonies_______ MicroScan identification panel: GLU +

RAF -

INO -

URE -

LYS +

TDA -

CIT -

TAR -

OF/G +

SUC -

RHA +

ADO -

H2S -

ARG -

ESC -

MAL +

ACE +

NIT +

SOR +

ARA +

MEL +

IND +

ORN +

VP -

ONPG +

CET no growth

DCB

Case 2:15 yr o male with urinary frequency and bloody urine. PAGE 4

Urine culture: catheter Mixed or pure culture?

Pure culture

Predominant?

N/A

Colony count for predominant CFU/ml

>100000 CFU/ml

Growth: BAP yes MAC yes BEA (if available)

Swarming round colonies Colorless swarming foul smelling colonies

Rule out potential pathogen – lactose fermenting organisms and gram positive organisms GS result

Gram negative rod

Oxidase

Negative

Final report: Proteus mirabilis with >100000 CFU/ml isolated

Appearance on MacConkey:__colorless – non lactose fermenting Colorless swarming foul smelling colonies ______ MicroScan identification panel: GLU +

RAF+

INO+

URE+

LYS-

TDA+

CIT+

TAR-

OF/G+

SUC+

RHA+

ADO-

H2S+

ARG-

ESC-

MAL+

ACE+

NIT+

SOR-

ARA+

MEL-

IND-

ORN+

VP-

ONPG+

CET no growth

DCB

Case 3 67 y o female present in ED with back pain and fever Urine culture: Clean catch Mixed or pure culture? Predominant?

Mixed Similar number of colonies seen

Colony count for predominant CFU/ml

>100000 CFU/ml

Growth: BAP yes MAC yes BEA (if available) NA

Big round grey colonies along with tiny dot like white colonies

Rule out potential pathogen GS result

Gram positive cocci, gram negative rods

Oxidase

Negative PAGE 5

Final report – No pathogen isolated. Mixed organisms suggestive of contaminants in the culture

Appearance on MacConkey: __lactose fermenting as well as NLF______ MicroScan identification panel: Not provided GLU

RAF

INO

URE

LYS

TDA

CIT

TAR

OF/G

SUC

RHA

ADO

H2S

ARG

ESC

MAL

ACE

NIT

SOR

ARA

MEL

IND

ORN

VP

ONPG

CET

DCB

Questions: 1. List three possible procedures for urine collection.  Clean catch midstream urine collection  Suprapubic needle aspiration of the bladder  Catheterization urine sample

2. Does urine have normal flora? No. it does not. urine is sterile and can have organisms in case of UTI

3. Describe how isolates are quantified on urine cultures: Calibrated loop of 1 ul (usually) should be used to quantify CFUs and streaking the sample down the center of the plate and then side to side across the plate without flaming the loop. The number of colonies counted is multiplied by 1000 which gives the CFU/ml. Normally colony count of 100 is considered to be significant

Section Three. Stool culture workup. Process:  Examine culture plates  Perform necessary tests to confirm or rule out a pathogen  Read MicroScan panel if necessary  Make your conclusion

PAGE 6

Activity: Case4: 30 yr o man presented to ED with vomiting and diarrhea. He reported eating potato salad 24 hours’ ago. Stool culture. Growth

Colonies description

BAP

Β hemolytic small grayish white colonies

MacConkey

Lactose fermenting small pink round colonies

MacConkey Sorbitol

Sorbitol fermenting pink small round colonies

XLD (or HE)

No growth

Define how you rule out potential pathogen: Salmonella

There is no growth in HE

Shigella

There is no growth in HE

E.coli 0157:H7

It is Non sorbitol fermenter

Vibrio cholera Final report

Microscan is not provided so the identity of organism cannot be determined Could be other species of E. coli

Note: No growth in CAMPY

Appearance on MacConkey: _ Lactose fermenting small pink round colonies ________ MicroScan identification panel: Not provided GLU

RAF

INO

URE

LYS

TDA

CIT

TAR

OF/G

SUC

RHA

ADO

H2S

ARG

ESC

MAL

ACE

NIT

SOR

ARA

MEL

IND

ORN

VP

ONPG

CET

DCB

Case 5: 5 yr o girl presented to ED with unexplained bruising and kidney pain. Mother reported that the patient had bloody diarrhea two days: Stool culture: Growth BAP MacConkey MacConkey Sorbitol

Colonies description Β hemolytic big round grayish colonies Lactose fermenter small round colonies with white dots in the centre Non sorbitol fermenter clear colonies PAGE 7

XLD (or HE)

No growth

Define how you rule out potential pathogen Salmonella

There is no growth in HE

Shigella

There is no growth in HE

E.coli 0157:H7

Non sorbitol fermenter clear colonies

Vibrio cholera Final report

E.coli 0157:H7 isolated

Appearance on MacConkey: __ Lactose fermenter small round colonies with white dots in the centre ________ MicroScan identification panel: Not provided GLU

RAF

INO

URE

LYS

TDA

CIT

TAR

OF/G

SUC

RHA

ADO

H2S

ARG

ESC

MAL

ACE

NIT

SOR

ARA

MEL

IND

ORN

VP

ONPG

CET

DCB

Case 6:35 y o female presented to ER with abdominal pain, bloody stool and fever. Stool culture: Growth BAP MacConkey MacConkey Sorbitol

Colonies description Small round grayish white colonies Non lactose fermenter small round colonies Sorbitol fermenting pink small round colonies

XLD (or HE)

Small blue/green colonies

Define how you rule out potential pathogen Salmonella Shigella E. coli 0157:H7

TDA+, VP+, Cit+, Lys+, H2S+ and growth in HE VP-, H2S-, LysIt is Non sorbitol fermenter

Vibrio cholera Final report

Salmonella isolated

Appearance on MacConkey: __ Non lactose fermenter small round colonies __________ MicroScan identification panel: PAGE 8

GLU+

RAF-

INO-

URE-

LYS+

TDA=

CIT+

TAR-

OF/G+

SUC-

RHA+

ADO-

H2S+

ARG-

ESC-

MAL-

ACE-

NIT+

SOR+

ARA-

MEL+

IND-

ORN+

VP-

ONPG-

CET no growth

DCB

Questions: 1. What is most likely the cause of the clinical symptoms in case 4? It can be Staphylococcus aureus or Bacillus cereus or Campylobacter. Microscan results need to checked and other testing to confirm the organism

2. Which organism can be confused with Salmonella sp on stool culture plates because of H2S production? Citrobacter can be confused with Salmonella spp 3. What is the most common mode of transmission for Vibrio sp.? Feco-oral route by contaminated food or water is the common route of transmission for Vibrio

4. What complication of the gastro intestinal infection most likelyis present in case 5? Since it is a case of infection by E. coli 0157:H7, it is likely to be Hemolytic uremic syndrome. Also known to cause acute hemorrhagic diarrhea

5. What additional enteric pathogen was not included in these stool cultures? What special growth conditions this pathogen require? Campylobacter is not included for which campy plate is used. In addition, Yersinia species is not included for which CIN agar should be used

6. Vibrio parahaemolyticus (a sometimes nasty side effect of eating raw oysters) grows well on routine media. But when a MicroScan panel is set up (0.5 McFarland standard suspension in water then diluted further in 25ml water used to inoculate the panel) it usually will not grow. Why? What can you do to get it to grow? pH of the medium is important for the growth of V. parahemolyticus so may be to change in the pH it was not able to grow. Using a selective media containing 1-7% NaCl would allow for its growth.

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