Growing Cultures

Part of Vaccines

Propagating pathogens and immune-stimulating materials in controlled conditions for vaccine preparation.

Why This Matters

To make a vaccine, you need a sufficient quantity of the target organism or its components. In nature, pathogens exist mixed with host tissue, competing organisms, and metabolic waste. Vaccine production requires isolating and multiplying the pathogen in controlled conditions — growing a pure culture in sufficient quantity to harvest, attenuate, and formulate.

This is microbiology at its most applied. The techniques for growing cultures were established by Pasteur and Koch in the 1870s-1880s and remain valid today. Broth cultures, solid media, serial dilution, colony isolation — these are skills that can be practiced with materials available in a technically capable rebuilding society.

Not all culture work requires the same infrastructure. Growing bacteria in broth needs boiling capability, glass containers, and simple nutrient media. Growing viruses requires living host cells (animal, embryonated eggs, or tissue culture) — far more demanding. This article focuses primarily on bacterial culture, which is more accessible.

Nutrient Media

Pathogens need nutrients to grow. The growth medium must supply these while being free of competing organisms (sterile).

For most bacteria, basic requirements are:

• Carbon source (glucose, peptone from meat or gelatin)
• Nitrogen source (protein hydrolysate, ammonium salts)
• Minerals (phosphate, sulfate, sodium, potassium)
• Water

Broth media (liquid): The simplest medium is meat broth — boil meat in water for 2-4 hours, filter to remove solids, add salt (0.5% sodium chloride), adjust pH to 7.0-7.4 with wood ash water (alkaline) or acidic plant extract, then sterilize by boiling or pressure steaming.

Gelatin/agar-solidified media: Adding a gelling agent to broth creates solid media where colonies grow as discrete spots, enabling isolation of pure strains.

• Gelatin: melts above 25°C, so it liquefies at room temperature in warm climates — limited use
• Agar: derived from seaweed; sets at ~42°C and melts at ~85°C; ideal for bacterial culture at body temperature

Agar preparation: extract from red algae (Gelidium, Gracilaria, or similar) by boiling in water, filtering, and allowing to set. Commercial agar is purified; home-prepared agar contains impurities but will grow most bacteria.

Sterilize all media by:

1. Boiling vigorously for 20+ minutes (kills most vegetative bacteria)
2. Pressure steaming (autoclave equivalent): 121°C for 15 minutes kills spores — achievable with a sealed pressure vessel
3. Filtration through fine porous material (does not kill but removes bacteria from heat-sensitive materials)

Inoculation and Incubation

Inoculating broth:

1. Obtain pathogen sample (tissue, wound exudate, lesion material, infected blood).
2. Transfer a small amount to sterilized cooled broth using a sterile instrument (heat a wire loop in flame until red-hot; let cool; dip in sample; transfer to broth).
3. Seal container with cotton plug or loose stopper to allow gas exchange while limiting contamination.
4. Incubate at appropriate temperature — most human pathogens grow best at 35-37°C.

Temperature maintenance: Without a thermostat, maintaining 37°C requires ingenuity:

• Water bath maintained at temperature with periodic addition of hot water, monitored with thermometer
• Insulated box with heated stones or bottles of hot water, replaced at intervals
• Incubation in clothing next to the body (body temperature)
• Near a heat source in a controlled environment

Signs of growth: Broth becomes turbid (cloudy) as bacteria multiply. Clear broth that remains clear after 48-72 hours at appropriate temperature suggests no growth. Turbidity should be confirmed as the desired organism by examining under a microscope if available.

Contamination signs: Unexpected colors, smells, or unusual turbidity patterns may indicate contamination with unwanted organisms. Discard and restart with improved sterile technique.

Serial Dilution and Isolation

To obtain pure cultures from mixed samples, use serial dilution and colony isolation.

Serial dilution in broth:

1. Prepare 10 tubes of sterile broth.
2. Add sample to tube 1; mix well.
3. Transfer 1/10 of tube 1 to tube 2; mix.
4. Transfer 1/10 of tube 2 to tube 3; continue.
5. Incubate all tubes. The last tube to show growth contains a dilution where individual organisms initiated separate cultures.

This approach helps when samples contain the desired organism at low numbers relative to contaminants — highly diluted cultures may contain only the target organism.

Plate isolation (if solid media available):

1. Liquefy agar medium by heating; cool to approximately 50°C (just above setting temperature — hot but just bearable to touch).
2. Add inoculum to liquefied agar; mix; pour into shallow flat container; allow to set (pour plate technique). Alternatively, streak cooled solid agar surface with inoculum (streak plate technique).
3. Incubate inverted (lid down) to prevent condensation from dripping onto colonies.
4. After 24-48 hours, discrete colonies appear — each represents growth from a single organism.
5. Transfer individual colonies to fresh sterile broth to obtain pure cultures.

Growing Viruses

Viruses cannot grow in lifeless nutrient media — they require living cells to replicate. This dramatically complicates culture work.

Embryonated egg culture: Fertilized chicken eggs incubated 7-12 days contain dividing cells on several membranes. Many viruses grow well in specific egg compartments.

1. Obtain fertilized eggs from a rooster-maintained flock.
2. Incubate at 37°C for 7-10 days, turning twice daily.
3. Candle the egg (hold over a light source in a dark room) to verify embryo viability — a living embryo shows blood vessels and movement.
4. Using sterile technique, drill a small hole through the shell at the appropriate location.
5. Inoculate a small volume of viral suspension into the desired compartment (allantoic cavity is most accessible).
6. Seal hole with wax; continue incubation.
7. After 2-5 days, harvest fluid containing virus.

This requires skill, temperature control, sterile technique, and a supply of fertile eggs. It is the simplest virus culture method available without full tissue culture.

Animal passage: Inoculating animals with viral material and harvesting tissues or fluids from infected animals is another approach. This was how early vaccines were produced — Jenner used cowpox from animal lesions directly.

Tissue culture (advanced): Maintaining living cells in nutrient solutions outside an animal allows virus growth without whole animals. This requires sterile solutions with specific nutrient composition, pH control, and temperature maintenance — significantly more complex but possible with developed laboratory infrastructure.

Quality Control in Culture

Sterility check: After producing a culture intended for vaccine use, always check for contamination:

• Take an aliquot of the culture and inoculate fresh sterile broth. After 48 hours, no growth should appear in the contamination check broth.
• Examine under microscope for unexpected organism types.

Identity confirmation: Use any available method to confirm you are growing the intended organism:

• Gram staining (requires basic stains): reveals bacterial shape and cell wall structure
• Colony morphology on solid media
• Biochemical tests (fermentation of specific sugars, enzyme tests)
• Clinical: inoculate a susceptible animal with the culture and observe expected disease signs

Quantity estimation: For consistent dosing, know approximately how many organisms you are working with:

• Turbidity comparison: dilute to match a known standard
• Counting under microscope in a defined volume
• Colony counts from dilution plating

These estimates need not be precise for early work but become essential when scaling up toward standardized vaccine production.

Safety During Culture Work

Growing concentrated pathogens is dangerous. Observe strict containment:

• Work in a dedicated area separate from food preparation, living space, and animal housing
• Wear covering clothing; protect wounds on hands
• Sterilize all waste immediately by boiling before disposal
• Never pipette by mouth
• Clean all work surfaces with phenol solution (0.5%) or dilute bleach after work
• If an accident occurs (spill, cut, or splash), clean immediately and apply antiseptic

The risk of inadvertent self-infection while culturing pathogens is real. Appropriate caution is required — but so is the work.

Topics covered in dedicated articles: Nutrient Media, Sterile Technique, Incubation