Unit 4: Total count and viable count determination - Subjective Questions

Total bacterial count refers to the number of bacterial cells present in a given volume or mass of a sample. In the spread plate technique, a measured volume of a suitably diluted sample is distributed over the surface of a solid agar medium using a sterile spreader. Each viable bacterial cell or cell cluster develops into a visible colony after incubation. The colonies are counted and used to estimate the number of bacteria in the original sample. The result is generally expressed as colony-forming units per millilitre, or , because one colony may arise from a single cell or a group of cells.

The following materials are commonly required:

• A well-mixed bacterial sample.
• Sterile physiological saline or another suitable diluent.
• Sterile dilution tubes or bottles.
• Sterile micropipettes or graduated pipettes with tips.
• Sterile Petri plates containing an appropriate nutrient agar medium.
• A sterile L-shaped glass or metal spreader.
• Alcohol for sterilizing the spreader.
• A flame source or suitable sterilization facility.
• Incubator maintained at the required temperature.
• Marker pen, labels, and a colony counter or magnifying device.
• Personal protective equipment, including a laboratory coat and gloves.

All materials must be sterile to prevent contamination and inaccurate colony counts.

Serial dilution is used to reduce the bacterial concentration to a level that produces countable colonies on an agar plate. A highly concentrated sample may produce a confluent lawn, making individual colonies impossible to distinguish. In contrast, excessive dilution may produce very few colonies and reduce the accuracy of the estimate.

A measured volume of the sample is transferred successively into tubes containing sterile diluent. Each transfer produces a known dilution factor. Several dilutions are plated so that at least one plate contains a suitable number of isolated colonies. Serial dilution therefore:

• Produces countable plates.
• Allows individual colonies to be distinguished.
• Improves the reliability of the calculated bacterial concentration.
• Reduces the effect of pipetting errors associated with handling very small volumes of concentrated samples.

The procedure is as follows:

1. Label sterile dilution tubes and agar plates with the sample identity and dilution factor.
2. Mix the original sample thoroughly to obtain a uniform suspension.
3. Prepare a series of decimal or other appropriate dilutions using sterile diluent.
4. Mix each dilution carefully before transferring a sample.
5. Pipette a known volume, commonly , onto the surface of a sterile agar plate.
6. Sterilize and cool the spreader, then distribute the inoculum evenly over the agar surface.
7. Allow the liquid to absorb into the agar.
8. Incubate the plates in an inverted position under suitable temperature and time conditions.
9. Select plates containing well-separated, countable colonies.
10. Count the colonies and calculate the bacterial concentration in the original sample using the dilution factor and plated volume.
11. Record the result as or , depending on the sample.

The bacterial concentration is calculated from the number of colonies obtained, the dilution plated, and the volume inoculated.

If is the number of colonies, is the dilution factor expressed as the reciprocal of the dilution, and is the volume plated in millilitres, then:

For example, if 86 colonies are obtained from a dilution and is plated:

The dilution correction accounts for the reduction in cell concentration, while division by the plated volume converts the count to a per-millilitre basis. If the original sample is measured by mass, the result may be expressed as instead.

A countable plate is a culture plate containing a number of well-separated colonies that can be counted with reasonable accuracy. In many teaching and routine laboratory procedures, a range of approximately 30 to 300 colonies per plate is considered acceptable, although the exact range may vary according to the standard method and organism.

This range is important because:

• Plates with fewer than about 30 colonies have greater relative statistical error.
• Plates with more than about 300 colonies may show crowding, merging, or inhibition of colony development.
• Well-separated colonies allow more accurate enumeration.
• Countable plates provide a more reliable estimate of the bacterial concentration in the original sample.

Plates showing confluent growth or spreading colonies should generally not be used for precise calculation.

Total count and viable count differ in the type of cells detected and the method used.

• Total count: Includes both living and dead bacterial cells. It may be determined by direct microscopic counting, electronic counting, or staining methods.
• Viable count: Measures only cells capable of multiplying under the specific culture conditions used. It is commonly determined by spread plate, pour plate, or membrane filtration methods.
• Unit of result: Total count may be expressed as cells per millilitre, whereas viable count is expressed as colony-forming units per millilitre, or .
• Interpretation: One colony may originate from one cell or a cluster of cells, so viable count is an estimate of viable cell units rather than an exact number of individual cells.
• Limitation: Cells unable to grow on the selected medium or under the incubation conditions are not detected by viable counting.

The spread plate method measures the ability of microorganisms to grow and form visible colonies under specified conditions. A colony may develop from:

• A single bacterial cell.
• A pair or cluster of cells.
• A chain or aggregate of cells.

Therefore, one visible colony does not necessarily represent one individual bacterial cell. In addition, some living cells may be stressed, dormant, or unable to grow on the selected medium. For these reasons, the result is reported as colony-forming units, or , rather than as an exact cell count. The term reflects the number of viable units capable of producing colonies under the particular culture conditions.

Use the formula:

where , , and .

Therefore, the estimated bacterial concentration in the original sample is .

Thorough mixing is necessary to distribute bacterial cells evenly throughout the sample. During storage or handling, cells may settle, attach to container walls, or form aggregates. If the sample is not mixed, the portion transferred to the plate may not represent the true bacterial concentration.

Proper mixing helps to:

• Produce a homogeneous suspension.
• Ensure that the aliquot plated is representative.
• Reduce variation between replicate plates.
• Improve the accuracy and reproducibility of the count.

Mixing should be vigorous enough to resuspend cells but should not create excessive aerosols or cause damage to fragile organisms.

Both techniques are used to estimate viable microorganisms, but they differ in procedure and colony location.

• Spread plate: A measured inoculum is placed on the surface of solid agar and distributed with a sterile spreader. Colonies develop mainly on the agar surface.
• Pour plate: A measured inoculum is placed in an empty sterile Petri dish and mixed with molten agar. Colonies develop both within the agar and on its surface.
• Inoculum volume: Spread plates usually require a relatively small volume, commonly , whereas pour plates may accommodate a larger volume.
• Temperature effect: Pour plating may expose sensitive organisms to warm molten agar, while spread plating avoids this exposure.
• Colony appearance: Surface colonies on spread plates are usually larger and easier to examine, whereas subsurface colonies in pour plates may be smaller.

Both methods require suitable dilution and countable plates for reliable enumeration.

The sterile spreader is used to distribute the inoculum uniformly across the surface of the agar. Uniform spreading increases the probability that individual bacterial cells will remain separated and form distinct colonies.

The spreader should be used as follows:

• Sterilize it by an approved method, such as flaming after moistening with alcohol, when appropriate.
• Allow it to cool before touching the inoculum or agar.
• Place the inoculum on the agar surface.
• Rotate the plate while gently moving the spreader over the surface.
• Avoid pressing heavily, which may damage the agar.
• Spread until the liquid is distributed evenly and absorbed.
• Re-sterilize the spreader between different samples or dilutions.

A hot spreader can kill bacteria, while an incompletely sterilized spreader can introduce contamination.

Important sources of error include:

• Inaccurate pipetting: Incorrect volumes directly affect the calculated concentration.
• Poor mixing: Uneven bacterial distribution produces unrepresentative aliquots.
• Incorrect dilution: Errors in preparing or labeling dilutions cause large calculation errors.
• Inadequate spreading: Uneven distribution may result in clustered colonies.
• Contamination: Contaminating organisms can increase the apparent count.
• Improper sterilization: A contaminated spreader or medium may produce false colonies.
• Unsuitable incubation: Incorrect temperature, time, atmosphere, or medium may prevent viable cells from growing.
• Counting errors: Merged, spreading, very small, or satellite colonies may be miscounted.
• Cell clumping: A cluster produces one colony and causes underestimation of individual cells.
• Use of unsuitable plates: Plates with too few or too many colonies provide unreliable estimates.

Using aseptic technique, replicate plates, appropriate dilutions, and standardized incubation reduces these errors.

Replicate plates are separate plates inoculated with the same dilution and volume. They are used to improve the reliability of the result by showing whether the counts are consistent.

Their importance includes:

• Detecting random pipetting or spreading errors.
• Identifying contamination on an individual plate.
• Allowing calculation of a mean colony count.
• Providing an estimate of variation between plates.
• Increasing confidence in the final bacterial concentration.

If replicate plates show widely different counts, the sample should be checked for inadequate mixing, inaccurate pipetting, uneven spreading, or contamination. Counts from acceptable replicate plates may be averaged before applying the dilution formula.

A confluent lawn is a continuous layer of bacterial growth in which individual colonies cannot be distinguished clearly. It usually occurs when the inoculum contains too many bacteria or when an insufficient dilution has been plated.

Such a plate should not be used for accurate enumeration because:

• Individual colonies cannot be counted reliably.
• Neighboring colonies may merge.
• Competition for nutrients may inhibit colony development.
• The apparent number of colonies may be lower than the actual number of viable units.
• Colony size and shape cannot be evaluated properly.

A higher dilution should be plated to obtain isolated colonies within the recommended countable range.

The plated volume must be included because the counted colonies represent only the bacteria present in that particular volume. The general equation is:

If is plated, the count is multiplied by to convert it to a per-millilitre value. If is plated, no volume correction is required. For example, 70 colonies from a dilution give:

For :

For :

Thus, failure to account for volume produces an incorrect result.

Important precautions include:

• Use sterile media, dilution tubes, pipette tips, and spreaders.
• Label every plate and dilution tube clearly before inoculation.
• Mix the original sample and each dilution thoroughly.
• Use calibrated pipettes and accurate sterile tips.
• Change the pipette tip between dilutions to prevent carryover.
• Allow a flamed spreader to cool before use.
• Spread the inoculum evenly without damaging the agar surface.
• Keep Petri plates open for the shortest possible time.
• Incubate plates in an inverted position to prevent condensation from spreading colonies.
• Include an uninoculated control plate when appropriate.
• Count only plates with well-separated colonies within the accepted range.
• Follow biosafety procedures and dispose of cultures using approved methods.

Incubation conditions determine which viable cells are able to multiply and form visible colonies. Important factors include:

• Temperature: The selected temperature should support the target organism. An unsuitable temperature may inhibit growth.
• Time: Insufficient incubation may result in colonies that are too small to see, while excessive incubation may allow colonies to merge or contaminants to appear.
• Atmosphere: Aerobic, anaerobic, or carbon dioxide requirements must be provided as needed.
• Medium composition: Nutrient availability, pH, salt concentration, and selective agents influence recovery.
• Moisture: Excess condensation can cause colonies to spread and merge.

Consequently, the count is not an absolute measure of all living cells. It represents cells able to grow under the chosen medium and incubation conditions.

Using the plate:

Using the plate:

The two estimates are reasonably close. The plate with 248 colonies lies within the commonly accepted countable range of approximately 30 to 300 colonies. The plate with 26 colonies is below that range and has a greater relative counting error. Therefore, the result may be reported as approximately , or as a suitable average if the laboratory protocol permits combining acceptable replicate results.

Contamination may be suspected when unexpected colony types appear, when an uninoculated control plate shows growth, or when replicate plates give inconsistent results. Contaminants may differ from the expected organism in colony colour, size, texture, margin, elevation, or pigmentation.

Contamination can be controlled by:

• Sterilizing media, glassware, spreaders, and other equipment.
• Working near an appropriate aseptic workstation.
• Minimizing the time plates and tubes remain open.
• Using fresh sterile pipette tips for each transfer.
• Avoiding contact between sterile materials and nonsterile surfaces.
• Properly flaming or otherwise sterilizing the spreader between samples.
• Including uninoculated controls.
• Correctly sealing, incubating, and handling plates.
• Discarding and repeating tests when contamination invalidates the count.