Stool Analysis Part 2: Microscopic Examination

Microscopic Examination of Stool

• Microscopic analysis of stool specimens includes examining the sample for:
  • Leukocytes
  • Epithelial cells
  • Blood
  • Qualitative fat
  • Parasites (eggs, larvae, cysts)
• A large number of leukocytes (> 50 cells per high power field) is typical of shigellosis
• A smaller number of pus cells (< 20 per high power field) is found in salmonellosis and invasive E. coli infections
• Few leukocytes (< 5 cells per high power field) are present in cholera, EPEC, and ETEC, and viral diarrhea

Epithelial Cells

• Normally, small to moderate numbers of epithelial cells are present in feces
• Large numbers of epithelial cells (or large amounts of mucus) indicate that the intestinal mucosa is scratched

Laboratory Diagnosis of Parasitic Infections

• The laboratory diagnosis of most parasitic infections is by demonstrating the ova of the parasite (stool ova and parasites test) in the infected person's stools
• Techniques used to examine the stool include:
  • Saline wet mount examination
  • Iodine preparation
  • Buffered methylene blue stain
  • Concentration techniques

Stool Examination (Saline Wet Mount and Iodine Staining) Method

• Gastrointestinal infestations (infections) by parasites are primarily diagnosed by detecting:
  • Live motile trophozoites (protozoans)
  • Cysts (inactive dormant stage of Protozoa)
  • Eggs and larvae (in case of Helminths) in stool
• Saline wet mount is used for the detection of:
  • Parasitic trophozoites and cysts of protozoa
  • Eggs and larvae of helminths
• Iodine staining method is used for the detection of:
  • Nuclei of some cyst forms of protozoans

Chemical Testing of Stool

• pH: Normal stool pH is 6-7.5
  • pH is acidic in amoebic dysentery
  • pH is alkaline in bacillary dysentery
• Occult Blood: Occult blood may be present in a variety of conditions, including malignancy of the gastrointestinal tract
  • Reagent used is benzidine powder
• Reducing Factors: Mono sugar and di sugar levels in stool (6mg/g) indicate disturbance in enzymes that digest sugar (e.g. lactase, sucrase)

Stool Culturing

• Certain bacteria are normally found in feces (the "normal flora" of the bowel)
• The presence of pathological types of bacteria may produce diarrhea and other signs of systemic infection
• Bacteria produce diarrhea in three main ways:
  • The organisms invade the intestinal wall, damaging tissue
  • The organisms produce toxins within the intestine that alter gastrointestinal motility
  • Toxins produced by bacteria are ingested (e.g. via foods) and produce diarrhea, although the organisms themselves are not detected in feces

Culture Media

• MacConkys Agar: Inhibits most of the gram-positive organisms between lactose fermenters and non-lactose fermenters
• Xylose Lysine Deoxycholate (XLD) Agar: Selective medium for the isolation of Salmonella and particularly Shigella from fecal samples
• Thiosulphate Citrate Bile Salt Sucrose (TCBS) Agar: Selective medium for the primary isolation of Cholerae
• Sorbitol MacConkys Agar: This medium is useful for screening 0157 E. coli

Culture Media Classification

• Based on consistency, culture media can be classified into:
  • Liquid media
  • Solid media
  • Semi-solid media

Cultivation

• Microbial culture is a method of multiplying microorganisms by letting them reproduce in predetermined culture media under controlled laboratory conditions
• The artificial medium should provide nutrients and a pH approximating those of tissues and body fluids

Purpose of Culturing

• Isolation of bacteria
• Properties of bacteria (i.e. culturing bacteria is the initial step in studying its morphology and identification)
• Estimate viable counts
• To test for antibiotic sensitivity
• To create antigens for laboratory use

Differential and Indicator Media

• Certain media are designed to recognize different bacteria based on their colony color
• Examples: MacConkey agar, Thiosulphate Citrate

Blood Agar

• Blood agar is considered differential because it is used to distinguish pathogenic bacteria based on the effect of bacterial enzymes known as hemolysins, which lyse red blood cells
• Blood agar contains a basal medium and 5-10% sheep, horse, or rabbit blood

Types of Hemolysis

• There are three types of hemolysis:
  • Beta hemolysis (β- hemolysis): Complete lysis or destruction of red blood cells
  • Alpha hemolysis (α- hemolysis): Partial lysis of RBC, resulting in greenish discoloration
  • Gamma-hemolysis (y-hemolysis): No lysis of RBCs

Microbiological Media

• Classifying Media:
  • Based on the nutritional component:
    • Simple (Basal) media
    • Complex (Enriched) media
  • Based on functional use or application:
    • Basal media
    • Enriched media
    • Selective media