7. Anaerobic Cultivation Methods

A. Classification of Organisms by Oxygen Tolerance

B. Specimen Collection and Transport

• Aspirate pus or fluid rather than swabbing (swabs expose organisms to air)
• Use anaerobic transport systems (pre-reduced transport tubes with oxygen-scavenging agents or CO₂/N₂ atmosphere)
• Deliver specimens to the laboratory as quickly as possible
• Never refrigerate anaerobic specimens; room temperature is preferred

C. Culture Methods

1. Anaerobic Jar (Evacuation-Replacement / GasPak System)

• Plates are placed inside a sealed jar
• Air is evacuated and replaced with a gas mixture (N₂, H₂, CO₂), or
• A GasPak envelope (hydrogen + CO₂ generator sachet) is activated inside a sealed jar; the hydrogen reacts with residual oxygen via a palladium catalyst to produce water, removing all O₂
• A methylene blue indicator strip turns white when O₂ is removed (normally blue in presence of O₂)
• Simple, economical, and effective for most clinical isolates

2. Candle Extinction Jar

• Plates and a lit candle are placed in a sealed jar; the candle burns out when O₂ is consumed
• Creates ~3-5% CO₂ and ~15% O₂ (not truly anaerobic)
• Used for capnophilic organisms (Neisseria, Haemophilus, Streptococcus pneumoniae) and microaerophiles - NOT for strict anaerobes

3. Anaerobic Chamber (Glove Box)

• A large, clear plastic, airtight chamber filled with an oxygen-free gas mixture of nitrogen, hydrogen, and carbon dioxide
• Specimens, plates, tubes, and instruments are introduced through an airlock (pass-through chamber) so the internal atmosphere is never disturbed
• Lab workers manipulate materials through built-in rubber gloves (hence "glove box")
• Allows continuous anaerobic processing: inoculation, subculture, reading plates - all done without ever exposing organisms to oxygen
• Offers the best recovery rates for strict obligate anaerobes and is essential for research-grade work

4. Roll-Tube Method (Hungate Technique)

• Molten agar is pre-reduced by boiling and cooling under N₂/CO₂
• Tubes are inoculated and rolled while cooling so agar solidifies in a thin layer lining the tube wall
• The tube headspace is maintained with anaerobic gas
• Originally developed for rumen microbiology; still used in research for extremely oxygen-sensitive organisms (e.g., methanogens, sulfate-reducing bacteria)

5. Thioglycolate Broth

• Liquid medium containing sodium thioglycolate, a reducing agent that lowers the redox potential
• Oxygen penetrates from the top, so only the lower portion of the broth is anaerobic
• Used as an enrichment broth and to demonstrate oxygen requirements (aerobic growth at top, anaerobic at bottom, facultative throughout)
• Not suitable as a sole culture method but useful as a backup or enrichment step

6. Pre-reduced Anaerobically Sterilized (PRAS) Media

• Media prepared and sterilized under anaerobic conditions (O₂ excluded throughout manufacture)
• Dispensed and stoppered under anaerobic gas mixtures
• Used with the roll-tube method or anaerobic chamber
• Superior recovery for fastidious strict anaerobes compared to conventionally prepared media

D. Incubation Conditions

E. Choosing the Right Method

F. Indicators and Quality Control

• Palladium catalyst pellets: Catalyze H₂ + O₂ → H₂O in jars; must be recharged (heated 160°C/2 hours) if contaminated
• Methylene blue strip: Colorless = anaerobic; blue = O₂ present (failure)
• Resazurin: Another redox indicator; pink = aerobic, colorless = reduced/anaerobic
• Control organisms: Clostridium sporogenes (ATCC 11437) used to validate anaerobic conditions

G. Media Used for Anaerobic Culture

• Brucella blood agar - most common general-purpose anaerobic plate
• Bacteroides bile esculin (BBE) agar - selective for Bacteroides fragilis group
• Phenylethyl alcohol (PEA) agar - inhibits gram-negative facultative bacilli; selects for anaerobic gram-positives and gram-negative anaerobes
• Kanamycin-vancomycin laked blood agar (KVLB) - selective for Bacteroides and Prevotella
• Cycloserine-cefoxitin fructose agar (CCFA) - selective for Clostridioides difficile

Source: Henry's Clinical Diagnosis and Management by Laboratory Methods, Culture Techniques chapter (ANAEROBIC BACTERIA section)

Agglutination Reactions - Principle & Applications

1. Definition

2. Basic Principle

Lattice Formation

• Stable, uniform particulates
• Pure antigen
• Specific antibody at an appropriate concentration

Role of Antibody Class

• IgM is far more efficient at producing complete agglutination than IgG, because of its pentameric structure (10 antigen-binding sites) and large size, allowing it to bridge the electrostatic repulsion between particles over greater distances.
• IgG antibodies, being smaller (bivalent), often cause incomplete agglutination (primary binding occurs but no visible clumping), especially when the particles carry a strong negative surface charge.

Zeta Potential (Electrostatic Repulsion)

• Lowering ionic strength of the medium (LISS - low ionic strength solution)
• Adding polymeric molecules: polymerized albumin (5-30%), dextran, polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), Polybrene
• Using an antiglobulin (Coombs) reagent

Prozone Phenomenon (Hook Effect)

3. Types of Agglutination Reactions

A. Direct (Active) Agglutination

• The antigen is naturally present on the surface of the particle (e.g., cell wall antigens on bacteria, surface antigens on RBCs).
• Antibody directly cross-links the particles.
• Examples:
  • ABO blood grouping (anti-A, anti-B antibodies agglutinate native RBCs)
  • Widal test (patient serum agglutinates Salmonella typhi H and O antigens)
  • Weil-Felix test (Proteus OX antigens used to detect rickettsial antibodies)
  • Cold agglutinin test

B. Passive (Indirect) Agglutination

• Soluble antigens are artificially coated onto an inert carrier particle (RBCs, latex, gelatin).
• Antibodies in the test specimen then agglutinate these sensitized particles.
• Examples:
  • TPHA / TPPA (Treponema pallidum antigen on tanned RBCs or gelatin particles - syphilis diagnosis)
  • Latex agglutination for Cryptococcus capsular antigen, Streptococcus grouping
  • Passive hemagglutination for various viral antibodies

C. Reverse Passive Agglutination

• Antibodies are coated onto the carrier particle instead of antigen.
• Used to detect soluble antigens in the specimen.
• Examples:
  • Detection of HBsAg (hepatitis B surface antigen)
  • Detection of hCG (pregnancy tests)
  • Detection of S. aureus toxins

D. Agglutination Inhibition

• A known antigen (in the test kit) coated on particles competes with free antigen in the specimen for a limited quantity of antibody.
• If free antigen is present in the specimen, it occupies the antibody and prevents agglutination of the coated particles → no clumping = positive test.
• Used for small haptens (drugs, hormones) that cannot cross-link on their own.
• Classic example: Original slide pregnancy test (inhibition of anti-hCG antibody by hCG in urine)

E. Coagglutination (Co-A)

• Uses protein A on Staphylococcus aureus Cowan I strain, which binds the Fc region of IgG antibodies, leaving the Fab (antigen-binding) ends free.
• Specific antibody is bound to the staph cells; when test antigen is added, visible agglutination occurs.
• Used for rapid identification of Streptococcus groups, Neisseria meningitidis serotypes, etc.

F. Antiglobulin (Coombs) Test - Indirect Agglutination

G. Hemagglutination Inhibition (HAI)

• Certain viruses (influenza, measles, rubella, mumps) can directly agglutinate RBCs by binding their surface glycoproteins.
• If the patient has specific antiviral antibodies, these block the virus from agglutinating RBCs → inhibition of hemagglutination = positive test.
• Example: HAI test for rubella immunity, influenza subtyping

4. Modern Formats

Latex Agglutination

• Latex particles (0.1-1 µm) coated with antigen or antibody
• Qualitative format: Mix a drop of sensitized latex with specimen on a black slide; read agglutination visually in 2-3 minutes
• Quantitative format: Measure turbidity (turbidimetry) or light scatter (nephelometry) in automated analyzers; sensitivity reaches sub-nanogram/mL level
• Applications: CRP, ASO, RF, Cryptococcus antigen, Group B Strep, hCG, bacterial CSF antigens

Gelatin Particle Agglutination

• Gelatin particles (~3 µm) have no antigenicity, reducing nonspecific reactions due to heterophilic antibodies
• Require much less serum dilution than erythrocytes
• Highly sensitive and specific
• Applications: HIV antibody screening, HBV, HCV, T. pallidum antibody (TPPA test)

Column (Gel) Agglutination Technology

• RBCs migrate through a dextran acrylamide gel column under centrifugation
• Agglutinated cells (large) are trapped at the top/middle of the column
• Unagglutinated cells (small, free) pellet at the bottom
• Stable, standardized endpoint; easily photographed for records
• Applications: Antibody screening, crossmatching, DAT in blood banking

Particle-Counting Immunoassay (PACIA)

• Counts the decrease in number of free unagglutinated particles after immune reaction using optical cell counter
• Sensitivity at nanogram/mL level

5. Clinical Applications Summary

6. Advantages and Limitations

Sources: Tietz Textbook of Laboratory Medicine, 7th Ed. (Agglutination Assays); Henry's Clinical Diagnosis and Management by Laboratory Methods (Principle of Particle Agglutination, Latex Agglutination, Gelatin Particle Agglutination, Column Agglutination Technologies)