Microbiology: Microorganisms, Staining, Culture, Specimen Collection & Immunity
Key Takeaways
In Gram staining, crystal violet is the primary stain, Gram's iodine the mordant, acetone or alcohol the decolorizer and safranin the counterstain; Gram-positive bacteria stay purple and Gram-negative bacteria appear pink.
Ziehl-Neelsen staining uses hot carbol fuchsin, an acid decolorizer and a methylene blue counterstain, so acid-fast bacilli such as Mycobacterium tuberculosis appear red on blue.
Blood cultures must be drawn before the first antibiotic dose, and urine for culture should reach the laboratory within about 2 hours or be refrigerated.
IgG is the only immunoglobulin that crosses the placenta, IgA protects mucosal surfaces and colostrum, IgM appears first in a primary response, and IgE mediates allergy.
Mantoux reactions and contact dermatitis are Type IV (delayed, T-cell) hypersensitivity, while anaphylaxis is Type I (IgE-mediated).
Microbiology explains why nurses wash hands, why a sputum sample is collected early in the morning, and why a blood culture is drawn before the first antibiotic. OSSSC lists it as a separate Annexure-II subject, and questions usually test staining, culture media, specimen handling and immunity.
1. Types of Microorganisms
| Group | Key features | Nursing examples |
|---|---|---|
| Bacteria | Prokaryotic cells with a cell wall; no nucleus; multiply by binary fission | Staphylococcus aureus, Escherichia coli, Mycobacterium tuberculosis |
| Viruses | Not cells; DNA or RNA in a protein coat; obligate intracellular parasites; unaffected by antibiotics | Hepatitis B, HIV, influenza, measles, rabies |
| Fungi | Eukaryotic; yeasts (single cells) or moulds (hyphae) | Candida albicans (thrush), Aspergillus, dermatophytes (ringworm) |
| Protozoa | Single-celled eukaryotic parasites | Plasmodium (malaria), Entamoeba histolytica (amoebic dysentery), Leishmania donovani |
| Helminths | Multicellular worms | Roundworm (Ascaris), hookworm, Wuchereria bancrofti |
Shapes and arrangements of bacteria
- Cocci (spherical): staphylococci in grape-like clusters; streptococci in chains; diplococci in pairs (pneumococci, meningococci, gonococci).
- Bacilli (rods): E. coli, Salmonella Typhi, M. tuberculosis.
- Comma-shaped (vibrios): Vibrio cholerae.
- Spiral forms: spirilla and spirochaetes (Treponema pallidum of syphilis, Leptospira).
Bacterial structure that matters clinically
- Cell wall: thick peptidoglycan in Gram-positive bacteria; thin peptidoglycan plus an outer membrane with lipopolysaccharide (endotoxin) in Gram-negative bacteria.
- Capsule: protects against phagocytosis (pneumococcus).
- Flagella: motility. Pili: attachment.
- Spores: highly resistant resting forms of Bacillus and Clostridium (tetanus, gas gangrene, C. difficile). Spores survive boiling and alcohol, which is why sterilization and soap-and-water hand washing matter.
- Growth curve: lag phase, log (exponential) phase, stationary phase, and decline (death) phase.
2. Staining Techniques
Gram staining (Hans Christian Gram, 1884)
- Crystal violet (primary stain) for about 1 minute.
- Gram's iodine (mordant) fixes the dye.
- Acetone or alcohol decolorizes for a few seconds.
- Safranin (counterstain).
Result: Gram-positive organisms keep the violet colour (Staphylococcus, Streptococcus, Clostridium). Gram-negative organisms lose it and take up the pink counterstain (E. coli, Klebsiella, Pseudomonas, Neisseria).
Ziehl-Neelsen (acid-fast) staining
- Strong carbol fuchsin, heated until steam rises.
- Acid (sulphuric acid or acid-alcohol) decolorizes everything except acid-fast cells.
- Methylene blue counterstain.
Result: acid-fast bacilli (AFB) appear red/pink against a blue background. The mycolic acid in the cell wall resists acid decolorization. This is the basis of sputum smear microscopy under NTEP.
Other stains to recognise: Albert's stain (metachromatic granules of Corynebacterium diphtheriae), India ink (capsule of Cryptococcus), and the KOH mount for fungal elements in skin or nail scrapings.
3. Culture Media
| Type | Purpose | Examples |
|---|---|---|
| Basal / simple | Supports non-fastidious organisms | Nutrient agar, peptone water |
| Enriched | Adds blood or serum for fastidious organisms | Blood agar, chocolate agar (Haemophilus, Neisseria) |
| Selective | Inhibits unwanted flora | Lowenstein-Jensen (M. tuberculosis), TCBS (V. cholerae), Wilson-Blair (S. Typhi) |
| Differential | Distinguishes colonies | MacConkey agar (lactose fermenters form pink colonies) |
| Enrichment (liquid) | Boosts small numbers of a pathogen | Selenite F broth (Salmonella) |
| Transport | Keeps organisms alive in transit | Cary-Blair (stool), Stuart's or Amies (swabs) |
M. tuberculosis grows slowly on Lowenstein-Jensen medium (weeks, not hours), which is one reason molecular tests such as CBNAAT and Truenat are preferred for rapid diagnosis.
4. Specimen Collection: Nursing Responsibilities
| Specimen | Key rules |
|---|---|
| Blood culture | Collect before antibiotics; strict skin antisepsis; usually two sets from two separate sites; inoculate the bottles without contaminating the cap; label at the bedside |
| Urine culture | Clean-catch midstream sample in a sterile container; from a catheter, aspirate through the sampling port, never from the drainage bag; deliver within about 2 hours or refrigerate at 4 °C |
| Sputum | Early-morning, deep-cough sample (not saliva) after rinsing the mouth with water; NTEP examines one spot and one early-morning sample |
| Stool | Fresh sample; for cholera or dysentery use a transport medium such as Cary-Blair |
| CSF | Send immediately at room or body temperature; never refrigerate (meningococci die in the cold) |
| Wound/throat swab | Swab the active margin or tonsillar area before antiseptic is applied; place in transport medium |
General rules: use sterile containers, follow standard precautions, collect before antimicrobials where possible, label with patient identifiers, date, time and source, and send with a complete request form. Delay and contamination are the commonest causes of misleading results.
5. Immunity and Hypersensitivity
Types of acquired immunity
| Type | Natural | Artificial |
|---|---|---|
| Active (own antibodies; long-lasting; slow onset) | Recovery from infection (e.g., measles) | Vaccines (BCG, MR, pentavalent) |
| Passive (ready-made antibodies; immediate; short-lived) | Maternal IgG across the placenta; IgA in colostrum | Immunoglobulins and antisera (rabies immunoglobulin, HBIG, anti-snake venom) |
Immunoglobulins
- IgG: most abundant; the only class that crosses the placenta; main antibody of the secondary response.
- IgA: secretions, tears and colostrum; protects mucosal surfaces.
- IgM: largest (pentamer); first antibody in a primary response, so IgM indicates recent infection.
- IgE: allergy and parasitic infections; binds mast cells.
- IgD: B-cell surface receptor.
Hypersensitivity reactions (Gell and Coombs)
| Type | Mechanism | Examples |
|---|---|---|
| I – Immediate | IgE on mast cells; histamine release | Anaphylaxis, allergic asthma, hay fever |
| II – Cytotoxic | IgG/IgM against cell-surface antigens | ABO transfusion reaction, Rh hemolytic disease of the newborn |
| III – Immune complex | Antigen-antibody complexes deposit in tissues | Serum sickness, post-streptococcal glomerulonephritis, SLE |
| IV – Delayed (cell-mediated) | Sensitised T-lymphocytes; 48–72 hours | Mantoux test, contact dermatitis, graft rejection |
Koch's postulates (the organism is found in every case, can be isolated in pure culture, reproduces the disease when inoculated, and is re-isolated) are the classic test of whether a microbe causes a disease.
During Gram staining of a wound swab, the organisms remain violet after the decolorizer is applied. What does this indicate?
The organisms are Gram-negative with an outer lipopolysaccharide membrane
The smear was over-decolorized and must be repeated
The organisms are Gram-positive with a thick peptidoglycan wall
The organisms are acid-fast bacilli
A nurse must send cerebrospinal fluid from a child with suspected meningococcal meningitis. How should the sample be handled?
Send it to the laboratory immediately, unrefrigerated
Refrigerate it at 4 °C until the laboratory opens next morning
Add formalin to preserve the organisms during transport
Freeze it so that the organisms cannot multiply in transit
Which immunoglobulin crosses the placenta and gives the newborn passive natural immunity?
IgM
IgE
IgG
IgA
Sections you finish are checked off in the contents.