Determination of cell count

/Determination of cell count
Determination of cell count2018-02-26T11:47:44+00:00

Determination of cell count

Determination of cell counts

Introduction

Many studies require the quantitative determination of bacterial populations. The two most widely used methods for determining bacterial numbers are the standard, or viable, plate count method and spectrophotometric (turbidimetric) analysis. Although the two methods are somewhat similar in the results they yield, there are distinct differences. For example, the standard plate count method is an indirect measurement of cell density and reveals information related only to live bacteria. The spectrophotometric analysis is based on turbidity and indirectly measures all bacteria (cell biomass), dead or alive. The standard plate count method consists of diluting a sample with sterile saline or phosphate buffer diluent until the bacteria are dilute enough to count accurately. That is, the final plates in the series should have between 25 and 250 colonies. Fewer than 25 colonies are not acceptable for statistical reasons, and more than 250 colonies on a plate are likely to produce colonies too close to each other to be distinguished as distinct colony-forming units (CFUs). The assumption is that each viable bacterial cell is separate from all others and will develop into a single discrete colony (CFU). Thus, the number of colonies should give the number of live bacteria that can grow under the incubation conditions employed. A wide series of dilutions (e.g., 10–4 to 10–10) is normally plated because the exact number of live bacteria in the sample is usually unknown. Greater precision is achieved by plating duplicates or triplicates of each dilution.

Requirements

  1. Bacterial culture
  2. Sterile normal saline
  3. Sterile petriplates
  4. Pipettes
  5. Nutrient agar

Equipments

  1. Laminar air flow
  2. Analytical balance
  3. Incubator

Procedure

  1. With a wax pencil, label the bottom of six petri plates with the following dilutions: 10–4, 10–5, 10–6, 10–7, 10–8, and 10–9. Label four bottles of saline or phosphate buffer 10–2, 10–4, 10–6, and 10–8.
  2. Using aseptic technique, the initial dilution is made by transferring 1.0 ml of liquid sample or 1 g of solid material to a 99-ml sterile saline blank. This is a 1/100 or 10–2 dilution. Cap the bottle.
  3. The 10–2 blank is then shaken vigorously 25 times by placing one’s elbow on the bench and moving the forearm rapidly in an arc from the bench surface and back. This serves to distribute the bacteria and break up any clumps of bacteria that may be present.
  4. Immediately after the 10–2 blank has been shaken, uncap it and aseptically transfer 1.0 ml to a second 99-ml saline blank. Since this is a 10–2 dilution, this second blank represents a 10–4 dilution of the original sample. Cap the bottle.
  5. Shake the 10–4 blank vigorously 25 times and transfer 1.0 ml to the third 99-ml blank. This third blank represents a 10–6 dilution of the original sample. Cap the bottle. Repeat the process once more to produce a 10–8 dilution.
  6. Shake the 10–4 blank again and aseptically transfer 1.0 ml to one petri plate and 0.1 ml to another petri plate. Do the same for the 10–6 and the 10–8 blanks.
  7. Remove one agar pour tube from the 48° to 50°C water bath. Carefully remove the cover from the 10–4 petri plate and aseptically pour the agar into it. The agar and sample are immediately mixed by gently moving the plate in a figure-eight motion while it rests on the tabletop. Repeat this process for the remaining five plates.
  8. After the pour plates have cooled and the agar has hardened, they are inverted and incubated at 35°C for 24 hours or 20°C for 48 hours.
  9. At the end of the incubation period, select all of the petri plates containing between 25 and 250 colonies. Plates with more than 250 colonies cannot be counted and are designated too numerous to count (TNTC). Plates with fewer than 25 colonies are designated too few to count (TFTC). Count the colonies on each plate. If at all possible, a special counter such as a Quebec colony counter should be used.
  10. Calculate the number of bacteria (CFU) per milliliter or gram of sample by dividing the number of colonies by the dilution factor. The number of colonies per ml reported should reflect the precision of the method and should not include more than two significant figures. For example, suppose the plate of the 10–6 dilution yielded a count of 130 colonies. Then, the number of bacteria in 1 ml of the original sample can be calculated as follows
    Bacteria/ml = (130) ÷ (10–6) = 1.3 × 108 or 130,000,000.
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