EXPERIMENT

1 Routine Examination of Stool

2 Isolation and Identification of Microorganisms from Stool Sample

INTRODUCTION

The Gastrointestinal (GI) tract hosts a large variety of microorganisms, commonly termed the
“normal microbial flora”. The number and type of organisms vary with the site. Escherichia coli is a
consistent normal microbial flora of the small intestine. Other enteric bacteria which may reside in
the small intestine include Klebsiella, Enterobacter, Proteus, Pseudomonas aeruginosa, and
Citrobacter. Although they constitute the normal flora many can act as opportunistic pathogens.
The lower intestinal tract, specifically the colon, hosts the greatest number of bacteria, the most
prevalent being Bacteroides. Other bacteria present in the large intestine include Bifidobacterium
(the "friendly" bacteria), and Clostridium (Clostridium perfringens , Clostridium difficile, Clostridium
tetani).

Gastrointestinal (GI) infections due to enteric pathogens may be in the form of gastroenteritis or
enterocolitis, and may be caused by bacterial, viral, fungal or parasitic agents. Clinical
manifestations may include nausea, vomiting, diarrhoea or dysentery and may be accompanied by
systemic effects depending on the causal agent. Bacteria commonly associated with GI infections
include Escherichia coli (ETEC, EPEC, EIEC, EHEC); Salmonella sp.; Campylobacter jejuni;
Shigella sp.; and Vibrio sp. Common viral agents encountered in GI infections include
rotaviruses; calicivirus; adenovirus 40 and 41; SRSV (small round structured viruses); astro
viruses; and echoviruses. Food-poisoning due to the ingestion of preformed exotoxin may be due
to Staphylococcus aureus, Clostridium perfringens, Bacillus cereus, or Clostridium botulinum.

Laboratory diagnosis due to bacterial aetiology is based on the isolation and identification of the
causal agent by culture methods, microscopy, biochemical tests, and serotyping. Stool is the
preferred clinical sample which should be collected preferably during active diarrhoea, or as soon
as possible after the onset of illness. Stool samples are collected in sterile, leak-proof, wide-mouth
containers. Rectal swabs may be taken from patients where stool collection is not feasible (eg,
infants or patients who cannot defaecate). Swabs are placed in an appropriate transport media
(Cary-Blair, Stuart, or buffered glycerol-saline) for transport to the laboratory.

Stool samples should be processed within 2 hours after collection. Stool should be plated directly
onto appropriate agar media for isolation and the isolates subsequently identified by microscopy,
biochemical, and serological tests.

Direct macroscopic and microscopic examination of stool may reveal parasites, neutrophils, and
red blood cells.

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OBJECTIVES

¾ To perform macroscopic and microscopic examination of stool specimen

¾ To culture provided stool sample/s on appropriate media
¾ To isolate and identify Proteus and/or Enterobacter spp. by appropriate biochemical tests
(To perform the API 20-E test)
¾ To perform serological typing for the Proteus or Enterobacter isolate
¾ To read and interpret the results obtained from the above procedures
¾ To convey the findings through written laboratory reports
¾ To observe the necessary laboratory precautions at all times

MATERIALS

1 Macroscopic and microscopic examination of stool

1.1 Sterile test tubes

1.2 Clean glass slides and coverslips
1.3 Pasteur pipettes
1.4 Bunsen lamp
1.5 Inoculating loop, stand
1.6 Clean microscopic glass slides
1.7 Lugol’s iodine
1.8 Sterile physiological saline

2 Isolation and identification of unknown organisms from stool

2.1 Stool samples spiked with Proteus mirabilis (labelled /coded PB-s)
2.2 Stool samples spiked with E. cloacae (labelled /coded EC-s)
2.3 Overnight culture of Proteus mirabilis on TSA (labelled /coded PB-t)
2.4 Overnight culture of E. cloacae on TSA (labelled /coded EC-t)
2.5 MacConkey, EMB (Eosin-Methylene Blue), and XLD agar plates
2.6 Nutrient agar slopes/slants
2.7 API 20-E test kit with interpretation chart
2.8 Sterile physiological saline (5 ml) in tubes
2.9 Light microscope, immersion oil, lens tissue, etc.
2.10 Gram stain set
2.11 Materials for oxidase test
2.12 Sterile mineral oil, 10% ferric chloride, Kovac’s reagent, VP reagent,
nitrate reduction reagents, 1.5 % hydrogen peroxide (for API 20-E test)
2.13 Ethanol
2.14 Forceps

3 Motility test

3.1 Depression slides and coverslips

3.2 Vaseline/paraffin wax

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 3.3 Sterile physiological saline

4 Serotyping

4.1 Clean glass slides and coverslips

4.2 Sterile Pasteur pipettes
4.3 Sterile physiological saline
4.4 Enterobacter or Proteus polyvalent “O” antiserum
4.5 Enterobacter or Proteus specific “O” antiserum

PROCEDURE

Day 1

1. Macroscopic and microscopic examination of stool sample

1.1 Macroscopic examination

¾ Examine for colour, consistency (formed, semisolid, fluid)

¾ Examine for presence of blood, mucous, undigested materials, parasites
¾ Determine pH (acid/alkaline)
¾ Record and report findings.

1.2 Microscopic examination of coverslip preparation/wet mount

¾ Clean glass slides free of grease.

¾ Place 1 drop of Lugol’s iodine and 1 drop of saline side by slide onto slide
using a sterile Pasteur pipette
¾ Emulsify the unknown stool sample provided
¾ Place a clean coverslip on each of the emulsified stool samples (hold
coverslip at 450 angle just touching the stool suspension, then bring it
down slowly/gently)
¾ Avoid creating air bubbles
¾ Examine first with 10X, then with 40X under reduced light
¾ Record and report findings

2. Stool for culture and identification of unknown organisms

2.1 Inoculation of MacConkey, Bismuth sulphite, and XLD agar

¾ Label MacConkey, EMB, and XLD agar plates as appropriate

¾ Inoculate a MacConkey, EMB, and XLD agar plates with the stool sample
provided
¾ Incubate at 370C

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 ¾ Examine for presence of growth after 24 and 48 hours

2.2 API 20-E test

¾ Labile the sterile physiological saline (5 ml) tube provided as appropriate

¾ Emulsify a colony from the provided TSA culture plate in 5 ml of sterile
physiological saline (mix well)
¾ Label and inoculate the API 20-E test strip (using a sterile Pasteur pipette)
according to the manufacturer’s instructions
¾ Incubate the API 20-E test (inoculated strip in incubation tray) at 370C

Day 2

1. Stool for culture and identification of organisms

1.1 Macroscopic examination of colony/colonies

¾ Examine the MacConkey agar plate for presence of growth

¾ Examine the EMB plate for presence of growth
¾ Examine the XLD agar plate for presence of growth
¾ Record/report findings including macroscopic features of the colonies

1.2 Microscopic examination of colony/colonies

¾ Make a smear of a representative NLF colony from MacConkey and XLD

agar plates
¾ Make a smear of a representative colony from EMB
¾ Stain by Gram’s method
¾ Examine under oil immersion objective
¾ Record/report findings

1.3 Motility test

¾ Label a clean depression slide as appropriate

¾ Prepare a hanging drop of a representative colony from cultures on
MacConkey, EMB, and XLD agar plate/s
¾ Examine for motility under 40X objective
¾ Record/report findings

1.4 Examination of API 20-E test

¾ Read reactions that do not need further test

¾ Perform the tests that are required according to the manufacturer’s
instructions and read the results
¾ Record/report findings

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 2. Serotyping

¾ Label a nutrient agar slant as appropriate

¾ Inoculate the nutrient agar slant with a colony from the TSA provided
¾ Incubate at 370C overnight

Day 3

1. Stool for culture and identification of organisms

1.1 Macroscopic examination of colony/colonies

¾ Read results of the plates after 48 hours of incubation

¾ Record and report findings

2. Serotyping

¾ Examine the nutrient agar slant for presence of growth

¾ Prepare clean, grease-free microscopic slide for serotyping
¾ Label as appropriate : “T” for test, “C” for control, and the code of the
culture
¾ Place 2 individual drops of physiological saline on the slide (use a sterile
wire loop each time)
¾ Emulsify colonies from the N/A slant in each drop of physiological saline
¾ Add a wire loopful of the Enterobacter /Proteus “O” polyvalent antiserum
to the “T” suspension
¾ Mix and examine for agglutination
¾ Repeat the serotyping steps for the Enterobacter /Proteus “O” specific
antiserum
¾ Record and report findings

RECORDING / REPORTING OF RESULTS

a) Macroscopic and microscopic examination of stool

Macroscopic appearance

Colour :
Consistency :
pH :
Presence of blood, parasites, others :

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 Microscopic appearance

RBC :
Pus cells :
Epithelial cells :
Ova of helminths :
Cysts/trophozoites of protozoa :
Bacteria :
Others :

b) Stool for culture and identification

Procedure/Test Findings Interpretation

Culture on Colony
MacConkey agar
Medium

Culture on EMB agar Colony

Medium

Culture on XLD agar Colony

Medium

Gram stain MacConkey agar :

EMB agar :

XLD agar :

Motility test MacConkey agar :

EMB agar :

XLD agar :

c) API 20-E test

Procedure/Test Findings Interpretation

API 20-E test Record findings in the form provided Interpret as appropriate
by the manufacturers

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d) Serotyping

Procedure/Test Findings Interpretation

Enterobacter /Proteus
polyvalent “O” antisera

Enterobacter /Proteus
specific “O” antisera

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