1. Mrs. Dalia Kamal Eldien Msc in Microbiology
Stool specimen
Mrs. Dalia Kamal Eldien
Msc in Microbiology
Lecture NO 9

2. Objectives Introduction Stool analysis tests Cause of diarrhea
Laboratory diagnosis of stool include stool for parasite examination& microbiological test (stool culture)

3. Introduction
 Proper gastrointestinal function is needed to eliminate toxic substances, pathogenic microbes, and undigested food particles from the body to prevent health problems.
A stool analysis: is a series of tests done on a stool (feces) sample to help diagnose certain conditions affecting the digestive tract .
These conditions can include infection (such as from parasites, viruses, or bacteria), poor nutrient absorption, or cancer .

4. Stool analysis include:
1-Fecal occult blood test can be used to diagnose many conditions that cause bleeding in the gastrointestinal system including colorectal cancer or stomach cancer
2-Chemical tests
Fecal PH: test may be used to determine lactose intolerance or the presence of an infection.
Fecal fat test: that checks for the male absorption of fat.
Fecal elastase: levels are becoming the mainstay of pancreatitis diagnosis.
3-Microbiology tests
4-Parasite testing

5. Microbiology & Parasitology tests
large numbers of abnormal bacteria, viruses, fungi, or parasites can grow in the intestines and cause infections and diseases. Although more than 50 different kinds of bacteria normally live in the intestines
Acute infective diarrhoea and gastroenteritis (diarrhoea with vomiting) are major causes of ill health and Loss of water and electrolytes from the body can lead to severe dehydration which if untreated can be rapidly fatal in young children, especially those that are malnourished, hypoglycaemic

6. Cause of diarrhoea
Invasive organisms such as shigellae, campylobacters, some salmonellae, and E. histolytica are associated with dysentery (passing of blood and mucus in stools).
non invasive organisms such as Rotaviruses, V. cholerae, and Entero toxogenic E. coli, cause watery (secretory) diarrhoea.
Diarrhoea is also associated with disease such as: HIV disease, malaria, severe malnutrition, pneumonia, hepatitis, cirrhosis of the liver, inflammation of the pancreas, tuberculosis of the intestine, colitis, previous surgery of the bowel, and malignant diseases of the intestinal tract.
Food poisoning also is another cause of diarrhoea

7. Possible pathogens Gram positive Gram negative
Clostridium perfringens Shigella species
types C Salmonella species
Clostridium difficile Campylobacter
Bacillus cereus (toxin) Yersinia enterocolitica
Staphylococcus aureus Escherichia coli (ETEC, EIEC, EPEC, VTEC)
Vibrio cholerae (01, 0139)
Other Vibrio species
Aeromonas species

8. VIRUSES:
Mainly Rotaviruses and occasionally Adenoviruses, Astrovirus, Calcivirus and Corona virus.
PARASITES
Entamoeba histolytica, Giardia lamblia, intestinal coccidia (Isospora, Cryptosporidium, Cyclospora) and other protozoan enteric pathogens

9. Laboratory diagnosis Specimen:
Faeces for microbiological examination should be collected during the acute stage of diarrhoea.
Give the patient a clean, dry, disinfectant-free suitable wide-necked container, Faecal material is placed into sterile container with the aid of the plastic spoon
The specimen should contain at least 5 g of faeces
Ask the patient to avoid contaminating the faeces with urine(Urinate before collecting).
Rectal swabs: Only when it is not possible to obtain faeces or from infants
Visible pus, mucus, or blood should be included in sample specimens.

10. Stool container with spoon

11. Labeling the specimen with patient Information (Name, age, department and diagnosis at admission )
Very importantly give a good history on the request form:
1. symptoms of gastroenteritis as bloody stool, vomiting, fever, abdominal pain and dehydration.
2.Food habits (drinking raw/unpasteruized milk, eating undercooked meat)
3. Exposure to farm environment
4. Medical disease as hepatitis, colitis, protozoal infection, and renal impairment.
5. Any medical therapy especially antimicrobial therapy

12. Criteria of specimen rejection
Specimen contaminated with urine.
Present of residual soap, or disinfectants.
Specimens received in grossly leaking transport containers
Dry specimens
Specimens submitted in fixative or additives

13. Macroscopical exam
Color of the specimen
Consistency: Whether it is formed, semi formed, unformed or fluid
Presence of blood, mucus or pus
Odor
Presence of worms seen in a freshly passed stool eg adult stages of Ascaris lumbricoides and Enterobius vermicularis
Normal faeces: Appear brown and formed or semi formed. Infant faeces are yellow-green and semi formed.

14. Microscopical exam
1-Saline and Eosin preparations:- to detect Entamoeba histolytica and other parasites
Place a drop of fresh physiological saline on one end of a slide and a drop of eosin stain on the other.
Using a piece of stick or wire loop, mix a small amount of fresh specimen (especially mucus and blood) with each drop Cover each preparation with a cover glass.
Examine the preparations using the X10 and X40 objectives :

15. Wet preparation

16. report the presence of :
 Large numbers of pus cells
 RBCs
 Amoebas, flagellates- cyst or trophozoite of E. histolytica trophozoites , orG. lamblia , or other parasite
 Eggs, larvae & cysts: Strongyloides larvae.
Using of Eosin 1%: this provide a pink background and that will help to clear the unstained objects.

17. Entamoeba histolytica cyst& trophozoite

18. Giardia lamblia cyst& trophozoite

19. Enterobius vermicularis egg (Pin worm)

20. Ascaris lumbricoides egg& worm

21. Hook worm egg

22. Taenia egg

23. Taenia saginata scolex& gravite

24. Scolex of Taenia solium with hooks and suckers

25. Fasciola hepatica

26. Schistosoma eggs

27. Continue stool exam 2-Basic fuchsin smear to detect campylobacters
Prepare when the specimen is unformed and, or, contains mucus, pus, or blood and is from a child under 2 years. Make a thin smear of the specimen on a slide. When dry, gently heat-fix. Stain by covering the smear with 10 g/l basic fuchsin for 10–20 seconds. Wash well with water and allow to air dry, use X100 Look for abundant small, delicate, spiral curved bacteria (often likened to gull wings), S-shapes

28. Campylobacters

29. 3-Motility test and 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 and darting motility. The preparation is best examined using dark-field microscopy
A Direct Gram stain is not useful beyond its determination of the presence of leukocytes, as it will not differentiate suspected pathogens from normal microbial flora.

30. Stool cultures
Routine stool cultures should be examined for the presence for Salmonella, Shigella, and Campylobacter species at minimum
Often cultures for Vibrio, Yersinia and E. coli O157:H7 are performed upon doctor request.
Enterohemorrhagic E. coli (EHEC) has been isolated from patients who have hemorrhagic colitis and hemolytic-uremic syndrome (HUS).

31. Inoculate the media making the first streak then use a sterile loop to streak for isolation. Non inhibitory media should always be inoculated first.
Routine stool culture
MacConkey agar
Hektoen enteric (HE) agar (or XLD or SS or DCA or bismuth sulphiteagar (BS) and brilliant green agar )
Skirrow& Butzler's (Selective agar for Campylobacter )
Gram negative(GN) broth or Selenite F – enrichment media enhances the growth of Salmonella and Shigella while suppressing the growth of normal bowel flora, they are subcultured to HE at 24 hours.

32. Yersinia culture
a. Cefsulodin-Irgasan-Novobiocin (CIN) agar or Yersinia Selective agar
b. Phosphate buffered saline (PBS) broth –as enrichment media suppresses the growth of normal bowel flora allowing easier detection of Yersinia species, but it also slows the growth of the Yersinia.
Vibrio culture
a. TCBS agar
b. Alkaline peptone broth as enrichment media

33. Incubate media
1. Temperature:
a. Campylobacter: 42ºC
b. PBS broth: 4ºC
c. CIN plate: room temperature
d. All other plates: 37ºC
2. Atmosphere:
a. Campylobacter: Microaerophilic (increased CO2)
b. All other plates: aerobically
3. Time: hours for all, 48 hours and 72 hours for Campylobacter

34. Colonial morphology
After 24 hours incubation, examine the HE and MAC plates for non-lactose fermenting colonies and H2S producing colonies.
These colony types are suspicious for Salmonella species and Shigella species, but may also be normal enteric flora – so biochemical screens must be performed to rule in or out the presence of these pathogens.
a. For colonies that are suspicious for Salmonella or Shigella perform KIA(kliglar iron agar) and LIA (lysine iron agar)
b. If screen is positive perform biochemical ID panels such as API 20 E.
c. Colonies that appear to be Salmonella species or Shigella species are confirmed by performing serological agglutination tests.

35. Salmonella sp. after 24 hours growth on XLD

36. Salmonella sp. On Hektoen enteric

37.  Shigella on XLD

39. A susceptibility test is performed only on any confirmed colonies of Salmonella and Shigella species when requested.
Treatment with antibiotics is not recommended for Salmonella species because it may induce a carrier state in the patient. In most case treatment of the clinical symptoms such as dehydration is sufficient.
If no suspicious colonies are found or if all biochemical screens are negative, the report is sent out as: No Salmonella or Shigella isolated

40. Serological Testing for Salmonella and Shigella
A. All isolates that biochemically resemble either Salmonella species or Shigella species must be confirmed by serological methods following site specific procedures.
B. For suspected Shigella species, test the following somatic (“O”) antigens:
Antigen A = Shigella dysenteriae
Antigen B = Shigella flexneri
Antigen C = Shigella boydii
Antigen D = Shigella sonnei
C. For suspected Salmonella species, test the following somatic (“O”) antigens:

41. Campylobacter
Colonies are tested for oxidase test. Any oxidase positive colonies are Gram negative stained to look for the typical curved rods of Campylobacter species. Sodium Hippurate test will confirm the species C. jejuni.
Susceptibility tests are not run on Campylobacter species because their resistance patterns are well established.
Macrolide antibiotics (Erythromycin, Clarithromycin, or Azithromycin) are the most effective agents against Campylobacter infection 

42. GULL WINGS

43. The special isolate:- Yersinia
After 24 hours incubation, examine the CIN plate for dark red colonies with a “bull’s eye” center surrounded by transparent border colonies that indicate mannitol fermentation and is suspicious for Yersinia. Confirm identification of suspicious organisms with various biochemical ID panels.
Vibrio
After 24 hours incubation, examine the TCBS agar for yellow colonies that indicate sucrose fermentation. Screen suspicious colonies biochemically.

44. yersinia enterocolitica on CIN agar

45. Vibrio on TCBS

46. Escherichia coli O157:H7
Setup stool culture with a MacConkey agar with 1% D-sorbitol (instead of lactose). After 24 hours of incubation, examine Mac-Sorbitol plate for colorless colonies (E. coli O157:H7 is sorbitol negative and most other normal flora strains of E. coli are sorbitol positive) Confirm identification of suspicious organisms with various biochemical ID panels and O157:H7 antisera.

48. For more reading
Textbook of Diagnostic Microbiology, Mahon & Manuselis, 3rd edition, Chapter 34, pages