SOP: Counting of Bacteria by Total Count and Viable Count

GIFSQ — Global Institute for Food Safety & Quality

Principle

A total count and a viable count measure different things. The total count, made by direct microscopic counting in a Helber chamber, counts every organism visible — including dead cells and cells indistinguishable from other particulate matter. The viable count assumes each living organism gives rise to one visible colony on plating; because bacteria in suspension often stay clumped, one colony may arise from one cell or from a clump of hundreds, so reproducible results depend on careful, uniform dilution and mixing.

Apparatus required

• Helber counting chamber (ruled area of 1 sq mm; 400 small squares, each 0.0025 sq mm; volume over each small square = 0.02 × 0.0025 mm³)
• Cover glass
• Inoculating loop
• Sterile pipettes
• Sterile test tubes with aluminium caps
• Water bath (45°C)
• Petri dishes
• Magnifier and tally counter, or colony counter
• Pen and ink (for marking colonies during counting)
• Pencil (for marking dilution numbers on dishes)

Reagents

a. 0.1% peptone water containing 0.1% Teepol and 0.1% methylene blue — for the total-count suspension; filter before use.

b. Formalin — a few drops added to the suspension.

c. Ringer solution, or preferably 0.1% peptone solution in water — the diluent for viable-count dilutions; if refrigerated, let it reach room temperature before use, to avoid cold-shock that may prevent organisms from reproducing. Flag: the book gives the preparation of Ringer solution in its “Saline and Ringer Solution” section; that preparation is not given in this chapter.

d. Nutrient agar or other suitable medium, tubed in 10-ml portions.

Procedure — Total counts

Add a few drops of formalin to the suspension and dilute it so that there are 5 to 10 organisms per small square of the chamber

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Place a loopful of the carefully mixed suspension on the ruled area of the chamber and apply the cover glass

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Allow 5 min for the bacteria to settle

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Count the number of organisms in 50–100 squares

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Divide the total number counted by the number of squares counted, to find the average number of organisms per square

Note — source flags: the printed line describing when the Teepol diluent applies (“if phase contrast or dark field is … for counting”) is illegible in the book extract, and the printed word describing the loopful placed on the chamber is illegible. Only the legible values above are carried.

Calculation — Total counts

Bacteria per ml = (Number of organisms per square) × (Dilution factor) × 20,000,000

Where,

• Number of organisms per square = average number counted in the small squares
• Dilution factor = the dilution of the suspension as plated
• 20,000,000 = organisms per ml corresponding to one organism per small square, from the printed chamber geometry (volume over each square 0.02 × 0.0025 mm³)

Note — source flag: the printed formula line is garbled (a stray “3” appears before 20,000,000). The value 20,000,000 is carried as derived from the printed chamber geometry, not from the garbled formula line itself.

Procedure — Viable counts: preparation of dilutions

Pipette 9 ml of sterile diluent (Ringer or 0.1% peptone solution) into each sterile test tube with an aluminium cap

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Mix the sample thoroughly by shaking; dip a sterile pipette about 0.5 in. into the suspension and remove 1 ml

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Deliver the 1 ml into the first dilution blank, about 0.5 in. above the level of the liquid; after 3 sec, blow out the pipette and discard it

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With a fresh sterile pipette, dip about 1 in. into the liquid, suck up and down ten times to mix, remove 1 ml, pipette it into the next dilution blank, and discard the pipette

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Repeat the process to obtain the required number of dilutions, numbering the dilutions serially: 1/10, 1/100, 1/1,000, 1/10,000, 1/100,000 (volume of original fluid per ml: 0.1, 0.01, 0.001, 0.0001, 0.00001)

Note — source flag: the printed third column of the dilution table (the 10⁻¹, 10⁻² … equivalents) is illegible in the book extract; the 1/10 … 1/100,000 dilutions and the 0.1 … 0.00001 ml per ml volumes are carried as printed.

Note: do not sterilize the diluent in test tubes with aluminium caps — volume changes may occur; sterilize it separately and dispense afterwards.

Procedure — Viable counts: plating and counting

Use two or more Petri dishes for each dilution and mark the dilution number on each dish with a pencil

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Melt the tubed nutrient agar (or other suitable medium) and cool to 45°C

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Pipette 1 ml of a dilution into the centre of a Petri dish, transfer the contents of one agar tube to the dish, and mix thoroughly — moving clockwise, counter-clockwise, back and forth and to-and-fro, six times each

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Allow the medium to set, invert the plate, and incubate for 24–48 hr

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Count the plates having 30–300 colonies, using a magnifier and a tally counter or a colony counter; while counting, mark the glass above each colony with pen and ink

Calculation — Viable counts

Colony count per ml = (Number of colonies per plate) × (Reciprocal of the dilution)

Where,

• Number of colonies per plate = colonies counted on the plate
• Reciprocal of the dilution = the inverse of the dilution plated (e.g. 10 for the 1/10 dilution)

Precautions

• Mix the sample thoroughly before and during the dilution series; clumps break unevenly and make results irreproducible.
• Never use refrigerated diluent cold — let it reach room temperature first, or cold-shock may stop organisms from reproducing.
• Count only plates with 30–300 colonies; crowded or nearly empty plates are unreliable.
• Use a fresh sterile pipette for every dilution transfer.

References

Ranganna, S., Handbook of Analysis and Quality Control for Fruit and Vegetable Products, 2nd edition.