MEASUREMENT OF MICROBIAL GROWTH – DIRECT METHOD

DIRECT MEASUREMENT OF MICROBIAL GROWTH 

1) Direct Microscopic Count 

• A specially designed slide called a Petroff-Hausser Cell Counter is also used in Direct Microscopic Count. Petroff-Hausser Cell Counter.
• In the Direct Microscopic Count method, a measured volume of a bacterial suspension is placed within a defined area on a Microscope slide.
• Because of time considerations, this method is often used to count the number of bacteria in milk. 
• A 0.01 ml sample is spread over a marked square centimeter of slide, stain is added so that the bacteria can be seen, and the sample is viewed under the oil immersion objective lens. The area of the viewing field of this objective can be determined.
• Once the number of bacteria has been counted in several different fields, the average number of bacteria per viewing field can be calculated. From these data, the number of bacteria in the square centimeter over which the sample was spread can also be calculated.

Petroff-Hausser Cell Counter

Advantages 

• No incubation time is required. 
• Holds for electronic cell counters, sometimes known as Coulter counters, which automatically count the number of cells in a measured volume of liquid. 
• Used in some research laboratories and hospitals.

Disadvantages

• Motile bacteria are difficult to count by this method.
• Dead cells are about as likely to be counted as live ones.
• High concentration of cells is required to be countable - about 10 million bacteria per milliliter.

2) Plate Count 

• The most frequently used method of measuring bacterial populations is the Plate count. 
• A Plate count is done by either the Pour plate method or the Spread plate method.
• In the Spread plate method, a volume (0.1 ml) of an appropriately diluted culture is spread over the surface of an agar plate using a sterile glass spreader. In the Pour plate method, a known volume (1 ml) of culture is pipetted into a sterile Petriplate. The usual practice, which is most valid statistically, is to count colonies only on plates that have between 30 and 300 colonies. Less than 30 colonies is represented as Too Least to Count (TLTC). More than 300 colonies is represented as Too Numerous to Count (TNTC). 

• Advantage - It measures the number of viable cells. 
• Disadvantage - It takes some time, usually 24 hours or more, for visible colonies to form. This can be a serious problem in some applications, such as quality control of milk, when it is not possible to hold a particular lot for this length of time.

3) Membrane Filter Technique

• The number of bacteria in aquatic samples is frequently determined from direct counts after the bacteria have been trapped on special Membrane filters.
• In the Membrane filter technique, at least 100 ml of water are passed through a thin Membrane filter whose pores are too small to allow bacteria to pass. Thus, the bacteria are filtered out and retained on the surface of the filter. This filter is then transferred to a Petridish containing a pad soaked in liquid nutrient medium, where colonies arise from the bacteria on the filter’s surface.
• Membrane filter technique is applied frequently to detection and enumeration of coliform bacteria, which are indicators of fecal contamination of food or water.

Membrane Filter Technique