Unit 4: Total count and viable count determination

I. Orientation

Total count and viable count are two related ways of estimating microorganisms in a sample. A total count includes cells whether living or dead, whereas a viable count measures cells capable of multiplying under the conditions used. The spread plate technique is primarily a viable-count method because each visible colony is assumed to arise from one viable cell or one viable cell cluster. Results are therefore expressed as colony-forming units per millilitre (CFU/mL) for liquids or CFU/g for solid materials.

• Governing principle: A measured volume of diluted sample is distributed over the surface of solid nutrient agar; viable microorganisms multiply into visible colonies during incubation.
• Counting unit: A colony represents one CFU, not necessarily one individual bacterial cell, because clumps and chains can produce a single colony.
• Dilution requirement: Samples are serially diluted so that at least one plate contains a countable number of colonies, commonly about 30–300 colonies.
• Aseptic convention: Sterile pipettes, spreaders, dilution blanks, and plates are required to prevent contamination from changing the count.
• Incubation condition: Temperature, time, atmosphere, and medium must be specified because they determine which organisms can grow.
• Calculation basis: The original concentration is calculated from colony number, dilution factor, and volume plated.
• Interpretive limitation: The result estimates organisms able to grow under the selected laboratory conditions, not every microorganism present in the original sample.

II. Total count of bacteria by spread plate technique — surface enumeration of viable bacteria

A. Purpose and principle

The spread plate method is used to estimate the concentration of viable, culturable bacteria in a sample by spreading a known volume across the surface of an agar plate. After incubation, separated colonies are counted and related mathematically to the original sample concentration.

• Purpose: To determine bacterial concentration in a sample such as water, broth culture, food homogenate, or environmental material.
• Growth basis: Each viable cell or cell aggregate deposited on the agar surface can divide repeatedly and form a visible colony.
• Surface distribution: A sterile L-shaped glass or disposable spreader distributes the inoculum across the agar surface rather than mixing it into the medium.
• Usual inoculum volume: Spread plating commonly uses 0.1 mL because a larger volume may remain pooled on the agar surface and a smaller volume increases counting error.
• Result type: The final value is reported as CFU/mL or CFU/g, depending on the original specimen.
• Underlying assumption: Colonies are sufficiently separated for individual counting, and each counted colony originated from viable material present in the plated dilution.

B. To determine total count of bacteria by spread plate technique

This procedure determines the bacterial count by serial dilution, surface inoculation, incubation, colony counting, and calculation of the original concentration.

• Materials: The essential materials are sterile nutrient agar plates, sterile dilution blanks, a micropipette or sterile pipettes, sterile tips, a sterile spreader, marker, disinfectant, incubator, and colony counter.
  • Medium: Nutrient agar or another appropriate solid medium supplies nutrients and a firm surface for colony development.
  • Diluent: Sterile physiological saline, phosphate-buffered saline, or peptone water maintains sample volume while reducing osmotic injury.
  • Sample: The test material must be mixed thoroughly before dilution so that bacteria are distributed as uniformly as possible.
• Aseptic preparation: Label plates before inoculation with sample identity, dilution, date, volume, and replicate number.
  • Example label: “Sample A, 10⁻⁵, 0.1 mL, plate 1” identifies the dilution and plated volume needed for later calculation.
  • Contamination control: Keep plates closed except during inoculation, use fresh sterile tips, and avoid touching the spreader or pipette tip to nonsterile surfaces.
• Serial dilution: Prepare successive tenfold dilutions by transferring 1 mL of sample into 9 mL of sterile diluent.
  • Dilution sequence: The first tube is 10⁻¹, the second is 10⁻², and a transfer from the 10⁻² tube into another 9 mL blank produces 10⁻³.
  • Mixing: Vortex or thoroughly invert each dilution before transferring from it; otherwise cells may settle and produce an inaccurate count.
  • Transfer technique: Use a new sterile pipette tip for every transfer to prevent carryover of bacteria from a higher concentration into a lower dilution.
• Plate inoculation: Transfer 0.1 mL of selected dilution onto the centre or upper surface of a properly dried agar plate.
  • Volume accuracy: A calibrated micropipette is preferred because the calculation directly depends on the plated volume.
  • Replicates: Inoculating two or three plates per dilution improves reliability and allows an obviously contaminated or damaged plate to be identified.
  • Plate order: Begin with the highest dilution and proceed toward lower dilutions when possible, reducing accidental contamination of low-count plates.
• Spreading the inoculum: Sterilize the spreader, allow it to cool, and move it gently over the agar surface while rotating the plate.
  • Sterilization: A glass spreader may be sterilized by dipping in alcohol and briefly flaming; flaming must be performed safely and only where permitted.
  • Distribution: The spreader should contact the liquid inoculum and distribute it over the entire surface without cutting or gouging the agar.
  • Absorption: Allow the inoculum to absorb before inverting the plate; excessive liquid can cause colonies to run together.
• Incubation: Invert the inoculated plates and incubate them under the specified conditions.
  • Inversion purpose: Inverting prevents condensation from dripping onto the agar and spreading colonies into indistinguishable streaks.
  • Typical teaching condition: Many nonfastidious bacteria are incubated at approximately 35–37°C for 24–48 hours, but the organism and medium determine the correct condition.
  • Recording condition: Temperature, duration, atmospheric condition, and medium should accompany the result because they affect recovery.
• Selecting countable plates: Count plates showing discrete colonies and an acceptable density, usually 30–300 colonies.
  • Too few colonies: Plates with fewer than about 30 colonies have greater relative counting error; they may still be useful when the sample is very dilute.
  • Too many colonies: Plates above about 300 colonies may show crowding or confluent growth, making individual colonies unreliable to distinguish.
  • Different dilution plates: Select plates from the same dilution when calculating replicates; if adjacent dilutions are both countable, use a consistent validated counting rule.
• Colony counting: Count every discrete colony, including colonies with different colour, size, shape, or opacity unless the protocol specifies selective counting.
  • Counting record: Record the dilution, plated volume, colony number, plate replicate, and any unusual appearance.
  • TNTC notation: “Too numerous to count” is used when colonies are confluent or exceed the laboratory’s accepted counting range.
  • Morphology: Distinct colony types may indicate mixed populations, but morphology alone does not identify bacterial species.
• Calculation: Convert the observed colony number into the concentration in the original sample using the dilution and plated volume.

CFU/mL = (Number of colonies × dilution reciprocal) / volume plated in mL

• Symbol definitions:
  • CFU/mL: Viable colony-forming units per millilitre in the original sample.
  • Number of colonies: Colonies counted on the selected plate.
  • Dilution reciprocal: Inverse of the dilution plated; the reciprocal of 10⁻⁵ is 10⁵.
  • Volume plated: Actual sample volume applied to the agar, expressed in millilitres.
• Worked example: If 86 colonies grow from 0.1 mL of a 10⁻⁵ dilution:

CFU/mL = (86 × 10⁵) / 0.1
       = 8.6 × 10⁷ CFU/mL

• Interpretation: The estimated original concentration is 86,000,000 viable CFU per millilitre, subject to the method’s assumptions and experimental error.
  • Replicate averaging: If replicate plates from the same dilution contain 84 and 88 colonies, use their mean, 86 colonies, in the calculation.
• Mean count: Averaging reduces random variation caused by uneven spreading or sampling.
• Agreement: Large differences between replicates suggest poor mixing, pipetting error, contamination, or uneven agar conditions.
  • Alternative calculation form: The same relationship may be written using the dilution factor.

CFU/mL = colonies × dilution factor × (1 / plated volume)

• Dilution factor: For a 10⁻⁵ dilution, the dilution factor is 10⁵.
• Volume correction: Plating 0.1 mL introduces a tenfold correction because the formula refers to one millilitre.
  • Controls: Control plates and dilution checks help distinguish bacterial growth from procedural contamination.
• Sterility control: Spread sterile diluent on an uninoculated agar plate; growth indicates contamination of the medium, diluent, spreader, or handling process.
• Media control: An uninoculated plate confirms that the agar itself was sterile before use.
• Positive control: A known culture can demonstrate that the medium and incubation conditions support expected growth, where included in the laboratory protocol.
  • Sources of error: Accuracy depends on representative sampling, complete mixing, precise pipetting, correct dilution tracking, and suitable incubation.
• Clumping: A bacterial cluster produces one colony, so the result may underestimate the number of individual cells.
• Uneven spreading: Incomplete surface distribution causes local crowding and an unreliable count.
• Viable but nonculturable cells: Cells that remain metabolically active but fail to grow on the chosen medium are not detected.
• Injured cells: Heat, drying, disinfectants, salt, or acidity may prevent damaged cells from forming colonies even though they were previously alive.
• Counting error: Colony overlap, satellite colonies, spreading organisms, and merged colonies reduce precision.
  • Advantages: The method is inexpensive, requires ordinary microbiology equipment, and produces isolated colonies that can be subcultured for further study.
• Quantitative usefulness: Serial dilution permits enumeration across a wide range of bacterial concentrations.
• Colony recovery: Individual colonies can be examined for morphology or transferred to fresh medium.
• Visual verification: Colony distribution provides an immediate check on whether the dilution series was reasonable.
  • Limitations: The spread plate does not measure all cells present and is not a direct microscopic total-cell count.
• Growth dependence: Only organisms able to grow on the selected agar under the selected conditions contribute to the count.
• Range restriction: Plates that are too crowded or too sparse produce less dependable estimates.
• Sampling limitation: A small plated volume may not represent a heterogeneous specimen.
• Terminological precision: Although the procedure is sometimes described as determining a “total bacterial count,” the measured value is specifically a viable culturable count expressed as CFU, not the total number of living and dead cells.
  • Safety and disposal: Treat unknown samples and cultures as potentially hazardous and follow institutional biosafety procedures.
• Personal protection: Use a laboratory coat, gloves, and eye protection when handling cultures, stains, disinfectants, or flame sources.
• Decontamination: Disinfect work surfaces before and after the procedure and sterilize or discard used materials according to laboratory policy.
• Waste handling: Place used plates, tips, pipettes, and spreaders in designated biohazard containers for appropriate decontamination.