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PROCESSING OF

STOOL

Outline
■ Possible pathogens

■ Commensal

■ Collection & transport of faeces

■ Lab examination of faeces

■ Summary

Possible pathogens
BACTERIA:
Gram positive Gram negative
1.Clostridium perfringens types A & C 1. Shigella species
2.Clostridium difficile 2. Salmonella serovars
3.Bacillus cereus (toxin) 3. Campylobacter sp.
4.Staphylococcus aureus 4. Yersinia enterocolitica
5. E.coli (toxin) (ETEC,
EIEC, EPEC, EHEC)
6. Vibrio cholerae 01, 0139
7. Other Vibrio species
8. Aeromonas species
■ Also M. tuberculosis

Possible pathogens
VIRUSES:
 Mainly rotaviruses & occasionally Norwalk agent, Adenoviruses,
Astrovirus, Calcivirus & Coronavirus

PARASITES:
 Entamoeba histolytica
 Giardia lamblia
 Intestinal coccidia (Isospora, Cryptosporidium, Cyclospora)
 Other protozoan enteric pathogens

Possible pathogens
■ Acute infective diarrhoea & gastroenteritis (diarrhoea + vomiting)~ ill
health & premature death in developing countries ~ CONTAMINATED
water supplies & POOR sanitation
■ Loss of water & electrolytes from the body ~ SEVERE DEHYDRATION
– if untreated can be rapidly fatal in young children, especially
those that are malnourished, hypoglycaemic & in poor health

■ Invasive Mo’s (Shigellae, Campylobacters, some Salmonellae & E.
histolytica) are associated with DYSENTERY (blood & mucus in stools)
■ Organisms such as Rotaviruses, V. cholerae & ETEC cause WATERY
(SECRETORY) DIARRHOEA

Possible pathogens ■ Diarrhoea may also be caused by: – Intestinal worms – Post-infective tropical malabsorption – Lactase deficiency – Antibiotic or other drug therapy which alters the intestinal NF – Dietary causes including gluten intolerance .

Possible pathogens ■ Diarrhoea is also associated with: – HIV disease – Malaria – Severe malnutrition – Pneumonia – Hepatitis – Cirrhosis of the liver – Inflammation of the pancreas – TB of the intestine – Colitis – Previous surgery of the bowel – Malignant diseases of the IT .

boydii & S. dysenteriae serotype 1 (Sd 1) is VIRULENT. S. cause at least 50% of the cases of bloody diarrhoea in young children in developing countries – S. sonnei) – Dysentery caused by Shigellae = BACILLARY DYSENTERY OR SHIGELLOSIS – WHO estimates that Shigella sp. (S. dysenteriae. causing endemic & epidemic dysentery with high death rates ■ It is highly infectious . Possible pathogens ■ Shigella sp. flexneri. S.

coli are common causes of enteritis in young children in developing countries . paratyphi cause enteric fever (typhoid & paratyphoid) which is endemic in many tropical & developing countries – Other Salmonellae cause food poisoning & bacteraemia ■ Campylobacter sp. jejuni & C. – C. – S. Possible pathogens ■ Salmonella sp. typhi & S.

‘rice water’ stools containing many vibrios are passed continuously~ urgent fluid replacement therapy to prevent collapse & death ■ V. Asia. vomiting. abdominal pain & acidosis ~ action of an exotoxin (cholera toxin) ~ causes water & electrolytes to flow into the bowel lumen – In severe infections. Possible pathogens ■ Vibrio sp. ■ V. America & Europe) . cholerae serogroups 01 (biotype El Tor) & 0139 cause endemic & epidemic cholera – Severe dehydration. parahemolyticus cause food poisoning (via contaminated seafood) in many parts of the world (Africa.

enterocolitica – Cause gastroenteritis in Africa. Possible pathogens ■ Strains of E. Europe & Canada – The organism is invasive & some strains are toxigenic . Japan. EPEC. EIEC & EHEC ■ Y. coli – Cause diarrhoeal disease – include ETEC.

Possible pathogens ■ Clostridium sp. perfringens type C ~ severe jejunitis (enteritis necroticans) or pigbel – cause of death in young children especially in Papua New Guinea. Bangladesh & some parts of East Africa – Infection is by the ingestion of contaminated pig meat – A lethal beta-toxin is produced ■ C. difficile ~ antimicrobial associated diarrhoea & sometimes PMC. ■ C. Solomon Islands. China. a rare & occasionally fatal condition . perfringens type A ~ food-poisoning by secreting enterotoxin in the intestine during sporulation – Alpha toxin is the main lethal toxin produced ■ C.

staphylococcal enterocolitis ~ complication of broad- spectrum antibiotic therapy ■ B. Possible pathogens ■ S. aureus food-poisoning – Ingestion of preformed toxin in contaminated food (often dairy products) – Occasionally. cereus food-poisoning – ingestion of preformed toxin usually in rice or other cereals which have been cooked & then stored for several days in warm temp .

Isospora & Microsporidia – Bacterial infections associated with diarrhoea in HIV/AIDS patients include Salmonella. Cyclospora. Possible pathogens ■ Rotavirus – Commonest cause of acute secretory diarrhoea in young children (6 months– 3 years) – Diarrhoea is the result of loss of extracellular fluid. due to impaired absorption – Diarrhoea & vomiting ~ severe dehydration – Most infections are accompanied by fever ■ Persistent diarrhoea – Often leads to diarrhoea-wasting syndrome (‘slim disease’) ~ common in AIDS – Due in part to opportunistic protozoal pathogens ~ Cryptosporidium. Shigella & Mycobacteria . Campylobacter.

Commensals ■ The NF of the GIT is greatly influenced by DIET ■ Mo’s which may form part of this NF include: – Coliform bacilli – Proteus – Pseudomonas – Clostridium – Bacteroides – Enterococcus – Lactobacilli – Also: ■ Mycoplasma ■ Candida sp. ■ A variety of protozoa & viruses .

Transfer a portion (about a spoonful) of the specimen. disinfectant-free bedpan or suitable wide-necked CONTAINER to pass a specimen ■ The container need NOT be sterile ■ Ask the patient to avoid contaminating the faeces with urine 2. Collection & transport of faeces ■ Faeces should be collected during the acute stage of diarrhoea In a hospital with a MB Lab 1. Give the patient a clean. pus or blood into a clean. dry. leak proof container . dry. especially that which contains mucus.

transfer to a separate container & send them to the lab for ID 3. Label the specimen & send it with a request form to reach the lab within 1 hr ■ If delay > 1 hr is anticipated. unformed or fluid. collect the specimen in Cary-Blair medium . worms. semi formed. or tapeworm segments are present 4. mucus. ■ Report also if blood. Write on the request form the colour of the specimen & whether it is formed. Collection & transport of faeces In a hospital with a MB Lab Cont’d Worms & tapeworm segments: ■ If present.

Insert the swab in the rectum for about 10 secs ■ Care should be taken to avoid unnecessary contamination of the specimen with bacteria from the anal skin Important: ■ When the specimen contains blood or amoebic dysentery is suspected. collect a specimen using a COTTON WOOL SWAB. Collection & transport of faeces In a hospital with a MB Lab Cont’d Rectal swabs: ■ Only when it is NOT possible to obtain faeces. deliver it to the lab ASAP – A fresh specimen is required to demonstrate actively motile amoebae & also to isolate shigellae .

Collection & transport of faeces In a health centre for transport to a MB Lab 1. Transfer a portion of the faeces to a cotton wool swab ■ Insert the swab in a container of STERILE CARY-BLAIR TRANSPORT MEDIUM . breaking off the swab stick to allow the bottle top to be replaced tightly . Request a specimen from the patient 2.

survive well in Cary-Blair medium ~up to 48 hrs ■ Campylobacter survive in Cary-Blair medium ~ up to 6 hrs ■ Merthiolate iodine formaldehyde (MIF) solution must NOT be used b/c MIF kills living organisms – MIF is used as a fixative for protozoal parasites ■ When CHOLERA is suspected: – Transfer about 1 ml of specimen into 10 ml of STERILE ALKALINE PEPTONE WATER & label – Specimen should reach the Lab within 8 hrs of collection . Shigella. Vibrio & Yersinia sp. Collection & transport of faeces In a health centre for transport to a MB Lab Cont’d ■ Salmonella serovars.

transfer these (using forceps) to a container of PHYSIOLOGICAL SALINE & send to the lab for ID . Collection & transport of faeces In a health centre for transport to a MB Lab Cont’d 3. Write a DESCRIPTION OF THE SPECIMEN on the request form ■ When worms or tapeworm segments are present.

Lab examination of faeces .

treated with rehydration & other supportive therapy w/o the need for antimicrobials & MB investigations ■ The MB examination of faecal specimens is mainly undertaken: – To investigate outbreaks of Dysentery (Shigellosis) . & other acute bacterial infective diarrhoeal disease of public health concern – To assist the central public health lab in the surveillance of endemic SS (including susceptibility of pathogens to antimicrobials) – To diagnose symptomatic Amoebic dysentery.Role of MB lab in investigating infective diarrhoeal disease ■ With most patients. diarrhoea is self-limiting . Giardiasis & other locally IMP intestinal parasitic infections . Cholera.

Lab examination of faeces: Day 1 1. semi-formed. Describe the appearance of the specimen ■ Colour of the specimen ■ Whether it is formed. ■ Enterobius vermicularis ■ Ascaris lumbricoides ■ Tapeworm segments – E. . Taenia sp. mucus or pus ■ Presence of worms: – E. unformed or fluid ■ Presence of blood.g.g.

EIEC dysentery.g. pale coloured. containing pus & mucus mixed with blood Shigellosis. often with blood & mucus Schistosomiasis Bloody diarrhoea (without pus cells) EHEC 0157 infection (haemorrhagic colitis) Watery stools ETEC. Cryptosporidiosis. Campylobacter enteritis Unformed with blood & mucus (acid pH) Amoebic dysentery Unformed or semi formed. unpleasant smelling Giardiasis. Rotavirus enteritis Rice water stools with mucous flakes Cholera Unformed/ watery & sometimes with blood. EPEC diarrhoea. Iron therapy . Appearance of faecal specimens in some diseases APPEARANCE POSSIBLE CAUSE Unformed. frothy. Hookworm blood) disease. other conditions that cause malabsorption stools that float on water (high fat content) e. mucus & Salmonella infection pus Unformed. post-infective tropical malabsorption Fluid stools (containing lactose with pH below 6) Lactase deficiency Unformed/ semi formed black stools (+ve occult Melaena (gastrointestinal bleeding).

Lab examination of faeces: Day 1 1. Describe the appearance of the specimen Cont’d ■ Blood can also be found in the stools of patients with: – Haemorrhoids – Ulcerative colitis – Tumors of the IT ■ Normal faeces: – Appear brown & formed or semi formed – Infant faeces are yellow-green & semi formed .

mix a small amount of fresh specimen (especially mucus & blood) with each drop – Cover each prep with a cover glass ■ The eosin prep must NOT be too thick otherwise it will NOT be possible to see AMOEBAE OR CYSTS . Examine the specimen microscopically SALINE & EOSIN prep to detect E. histolytica & other parasites ■ Place a drop of fresh PHYSIOLOGICAL SALINE on one end of a slide & a drop of EOSIN STAIN on the other – Using a piece of stick or wire loop. Lab examination of faeces: Day 1 2.

lamblia trophozoites – Motile Strongyloides larvae – The eggs & cysts of parasitic pathogens . Look especially for : – Motile E. Lab examination of faeces: Day 1 2. Histolytica trophozoites containing red cells – Motile G. histolytica & other parasites Cont’d ■ Examine the prep using t X10 & X40 objectives with the condenser iris closed sufficiently to give good contrast. Examine the specimen microscopically SALINE & EOSIN prep to detect E.

Examine the specimen microscopically : ADDITIONAL Methylene blue prep to detect faecal leucocytes when the specimen is unformed ■ Place a drop of MB stain on a slide. Lab examination of faeces: Day 1 2. Mix a small amount of specimen with the stain & cover with a cover glass ■ Examine the prep for faecal leucocytes using the X40 objective with the condenser iris closed sufficiently to give good contrast ■ Report also the presence of RBC as these are often present with pus cells in inflammatory invasive diarrhoeal disease .

Examine the specimen microscopically : ADDITIONAL Methylene blue prep to detect faecal leucocytes when the specimen is unformed Faecal leucocytes (WBCs): ■ Look for mononuclear cells & Polymorphonuclear cells (pus cells) ■ Mononuclear cells contain a nucleus which is NOT LOBED whereas Polymorphonuclear cells contain a nucleus which has 2 OR MORE LOBES ■ Sometimes the cells are too damaged to be recognized (do not attempt to identify) ■ Pus cells ~associated with bacteria that cause inflammation of the large intestine ■ Often red cells are also found ■ Mononuclear cells ~ found mainly in typhoid & in some parasitic infections (amoebic dysentery) . Lab examination of faeces: Day 1 2.

difficile 4. Shigella species 2. C. perfringens (causing pigbel) 5. C. Aeromonas species . E. histolytica 5. Campylobacter species 3. B. coli 2. Lab examination of faeces: Day 1 Causes of inflammatory diarrhoeal disease: 1. EIEC Less common: 1. Salmonella (non-typhoid serovars) 4. enterocolitica 3. Y.

enteritis . gently heat-fix. ■ Examine the smear for Campylobacters using the X100 oil immersion objective. S-shapes & short spirochaetal forms ■ Examination of stained faecal smears for Campylobacters ~sensitive method for presumptive diagnosis of C. or blood & is from a child under 2 yrs ■ Make a thin smear of the specimen on a slide. delicate. Wash & allow to air dry – 10 g/l Basic fuchsin: Dissolve 1 g basic fuchsin in 100 ml of water & filter. Look for abundant small. When dry. spiral curved bacteria (gull wings). Lab examination of faeces: Day 1 2. Examine the specimen microscopically : ADDITIONAL Basic fuchsin smear to detect Campylobacters ■ Prepare when the specimen is unformed & or contains mucus. Stain by covering the smear with 10 g/l basic fuchsin for 10–20 secs. pus.

cholerae ■ Examine also a Gram-stained smear of the culture for GN vibrios (use 1 in 10 dilution of carbol fuchsin as the counterstain instead of neutral red) . Examine the specimen microscopically : ADDITIONAL Motility test & Gram stained smear when cholera is suspected ■ Examine an ALKALINE PEPTONE WATER CULTURE (sample from the surface of the culture) for vibrios showing a rapid & darting motility ■ The prep is best examined using DF microscopy but the vibrios can also be seen using transmitted light ■ Experience is required to identify the characteristic motility of V. Lab examination of faeces: Day 1 2.

make a thick suspension of it in about 1 ml of sterile peptone water Xylose lysine deoxycholate (XLD) agar ■ Inoculate a loopful of fresh emulsified faeces or a fluid specimen on XLD agar ■ Incubate the XLD agar plate aerobically at 35–37 °C overnight. Culture the specimen ■ When the specimen is formed or semi formed. . Lab examination of faeces: Day 1 3.

sonnei strains) ■ Salmonellae also form PINK-RED COLONIES even though they ferment xylose with acid production – B/c they break down LYSINE which gives an alkaline rxn – H2S producing salmonellae form RED COLONIES WITH BLACK CENTRES ■ Some Proteus & Edwardsiella sp. Lab examination of faeces: Day 1 3. or sucrose (except some S. lactose. Enterobacter sp. & some other Enterobacteria produce YELLOW COLONIES~ CHO fermentation ■ Less selective medium (MAC) maybe used in addition to XLD agar . Culture the specimen ~ XLD agar: ■ Selective medium used for isolation of S&S from faecal specimens ■ Contains the indicator PHENOL RED (red ~ alkaline pH & yellow ~acid pH) ■ Shigellae form PINK-RED COLONIES b/c they DONOT ferment xylose. form PINK-RED COLONIES WITH BLACK CENTRES ■ E. coli.

Culture the specimen ~ XLD agar: E.coli Salmonella Mixed culture of E. Lab examination of faeces: Day 1 3.coli & Salmonella Shigella .

of vibrios (e. cholerae colonies of E. Culture the specimen Uninoculated ADDITIONAL Alkaline peptone water & TCBS agar when cholera is suspected ■ Inoculate several loopfuls of specimen in alkaline (pH 8. – Alkaline peptone water is a useful transport Much smaller .6) peptone water & incubate at 35–37 °C for 5–8 Large. faecalis ■ Subculture several loopfuls of the peptone water culture (taken from the surface) on thiosulphate citrate bile-salt sucrose (TCBS) agar & Incubate aerobically at 35–37 °C o/n . yellow colonies of Vibrio cholerae hrs – Prior enrichment in alkaline peptone water is NOT necessary if the specimen is likely to contain large No. yellow medium for V.g. in acute cholera). Lab examination of faeces: Day 1 3.

coli 0157 is suspected ■ Inoculate a loopful of specimen on sorbitol MacConkey agar & incubate the plate aerobically at 35–37 °C o/n Sorbitol MacConkey agar ■ Contains the CHO SORBITOL instead of lactose. when an outbreak of E. Lab examination of faeces: Day 1 3. coli 0157 . ■ E. coli 0157 ~ colourless colonies ~ DOES NOT ferment sorbitol ■ Most other E. coli strains & other Enterobacteria~ pink colonies ~ ferment sorbitol ■ A useful way of screening for E. Culture the specimen ADDITIONAL Sorbitol MacConkey agar.

. Culture the specimen ADDITIONAL Blood agar & Improved Preston blood-free medium . Lab examination of faeces: Day 1 3. droplet-like colonies ■ Examine colonies microscopically for Campylobacters & perform an oxidase test. jejuni & C. when Campylobacter is suspected ■ Inoculate a loopful of specimen on BA/ Preston medium & incubate the plate at an microaerophilically (atm of reduced O2 (5–10%) with added CO2 (about 10%)) at 35–37 °C o/n Blood agar ■ C. coli produce non-haemolytic spreading.

. – A swarming growth may occur ■ Examine the colonies microscopically & perform an oxidase test. moist. Culture the specimen ADDITIONAL Blood agar & Improved Preston blood-free medium . flat-spreading colonies ■ Some strains ~ green hue or a dry appearance with or w/o a metallic sheen ■ C. when Campylobacter is suspected Improved Preston blood-free medium: ■ C. coli ~ produces creamy-grey. slightly raised colonies. moist. Lab examination of faeces: Day 1 3. jejuni ~ grey.

strains of S. Providencia & Pseudomonas ~ red colonies ■ Some Proteus strains (are also H2S producing ) ~ red colonies with black centres MacConkey agar ■ Shigellae & Salmonellae & other NLF organisms ~colourless colonies ■ E. – Shigella & H2S–ve strains of Salmonella ~ 1–2 mm dia red colonies – H2S +ve Salmonellae (e. typhimurium)~ Red colonies with black centres ■ Proteus. coli & other LF ~ produce pink colonies . Examine and report the cultures XLD agar culture ■ Look for colonies that could be Shigella or Salmonella. Lab examination of faeces: Day 2 & Onwards 4.g.

No further tests are required . Lab examination of faeces: Day 2 & Onwards 4. Examine and report the cultures ID of suspect S&S isolates ■ Perform a urease test using urea broth – A +ve urease test within 2–4 h indicates that the organism is probably Proteus.

stab first the butt & then streak the slope. ■ Close the tube with a cap & incubate at 35– 37 °C o/n . Lab examination of faeces: Day 2 & Onwards 4. proceed as follows: 1. Inoculate a tube of Kligler iron agar ■ Use a sterile straight wire. Perform indole & lysine decarboxylase tests 2. Examine and report the cultures ID of suspect S&S isolates ■ Perform a urease test using urea broth – When the urease test is -ve at 4 hrs.

Red (Alkaline rxn) Yellow (Acid rxn) . Red (Alkaline rxn) Yellow (Acid rxn) . + Red (Alkaline rxn) Yellow (Acid rxn) +* + S. . flexneri . + Red (Alkaline rxn) Yellow (Acid rxn) + (Weak) - Other -* . Typhi + . -* S. Tests used to identify presumptively S&S KIA Medium Reactions Motility Indole LDC Slope Butt Black Cracks (H2S) (Gas) SHIGELLAE S. D . -* S. + Red (Alkaline rxn) Yellow (Acid rxn) +* D Salmonella serovars D= different strains give different results. dysenteriae . paratyphi A + . Red (Alkaline rxn) Yellow (Acid rxn) . paratyphi + . - S. + Red (Alkaline rxn) Yellow (Acid rxn) + + S. D . Red (Alkaline rxn) Yellow (Acid rxn) -* + S. - SALMONELLAE S. boydii . . Red (Alkaline rxn) Yellow (Acid rxn) . . paratyphi B + . D . * A few exceptions . sonnei .

typhi ~ small amount of blackening & NO cracks in the medium . Examine and report the cultures RESULTS LDC ■ Shigella are LDC -ve. Other shigellae give variable indole rxn. Salmonella serovars are indole -ve. KIA ■ SS ~ pink-red slope & yellow butt ■ Many Salmonellae ~produce blackening (H2S production) & cracks (gas production) from glucose fermentation ■ S. Paratyphi A which is LDC -ve. Indole ■ S. sonnei is indole -ve. Salmonella serovars are LDC +ve except S. Lab examination of faeces: Day 2 & Onwards 4.

– Examine for agglutination against a dark background. – When there is agglutination (autoagglutination). add one loopful of test antiserum & mix.Lab examination of faeces: Day 2 & Onwards 4. – A NA culture should be sent for further testing to a Reference Lab ■ When there is NO autoagglutination. Examine and report the cultures Serological ID of SS ■ Test any isolate giving rxns suggestive of SS using a slide technique – Emulsify a small amount of growth from the KIA culture in a loopful of physiological saline on a slide – Mix by tilting the slide backwards & forwards for about 30 secs. ■ A +ve test will show strong clear agglutination within 1 min . the strain is unsuitable for serological testing. examine for agglutination.

& enterococci produce small yellow colonies ■ Proteus strains may produce yellow or yellow-green colonies with black centres ■ Some Pseudomonas strains form small green colonies. cholerae is sucrose fermenting & produces yellow 2–3 mm in dia shiny colonies with a yellow colour in the medium – With prolonged incubation (48 h or >) the colonies may become green ■ V. parahaemolyticus is non-sucrose fermenting ~ green-blue 2–3 mm in dia colonies ■ V. fluvialis ~ sucrose fermenting ~ may occasionally be isolated as a pathogen ■ V. . Examine and report the cultures: TCBS agar culture ■ V. Aeromonas sp. mimicus~ non-sucrose fermenting ~ green-blue colonies & is sometimes isolated Selectivity of TCBS ■ Very occasionally. Lab examination of faeces: Day 2 & Onwards 4.

■ Perform an oxidase test on the NA culture – It is not possible to perform an oxidase test directly from a TCBS culture b/c the acid produced by the sucrose fermenting colonies will inhibit the oxidase rxn – Sub culturing to nutrient agar is also required to perform serological tests reliably. ■ Note: When the oxidase test is +ve. presume the isolate to be V. Lab examination of faeces: Day 2 & Onwards 4. cholerae isolate ■ Examine a Gram stained smear of the culture for Gram negative vibrios – The organisms may appear less curved after culture. Examine & report the cultures Identification of a suspect V. cholerae – Tested serologically (using NA culture) to confirm the organism is V. ■ Subculture the organism on a slope of NA (use a heavy inoculum) & incubate for 4–6 hrs. cholerae 01 or 0139 .

Describe Specimen Report – Colour – Whether formed. Culture XLD agar Alkaline peptone water & TCBS agar: Specimen Incubate aerobically When cholera is suspected MacConkey agar Sorbitol MacConkey agar Incubate aerobically When infection with VTEC 0157 is suspected .Examine Saline and eosin: Methylene blue: Microscopically To detect parasites To detect WBCs when the specimen is unformed Basic fuchsin smear: When Campylobacter enteritis is suspected Motility test & Gram smear: From alkaline peptone water culture when cholera is suspected 3. Summary of the MB Examination of Faecal Specimens DAY 1 ADDITIONAL INVESTIGATIONS 1. semi formed. unformed. pus – Presence of worms 2 . fluid – Presence of blood. mucus.

Summary of the MB Examination of Faecal Specimens DAY 2 & ONWARDS ADDITIONAL INVESTIGATIONS Examine & XLD & MAC Cultures Examine TCBS culture Report Look for SS: -Look for colonies that could be Cultures – Exclude Proteus using urease test V. cholerae – Identify presumptively: –Gram stain colonies ● LDC & indole – Subculture on nutrient agar ● Motility – Perform oxidase test ● KIA – Identify serologically – Identify serologically Examine sorbitol MacConkey – Perform susceptibility testing on agar culture Shigella isolates – Look for colourless colonies that could be EHEC – Perform 0157 latex agglutination test .