Title 10. COUNTING OF BACTERIA -TOTAL AND VIABLE COUNTING TECHNIQUES.pdf ✅

PHARMD GURU Page 1 COUNTING OF BACTERIA Measurement of microbial growth and physiology is an important parameter of microbial study. This measurement to a great extent depends on the number of microorganisms present in a culture. Growth of microorganisms can be quantitatively measured using various techniques. A) DETERMINATION OF CELL NUMBER: 1) Total count / direct methods: a) Direct microscopic count / Breed method. b) Counting chamber method / Haemocytometer method. c) Proportional count method. d) Electronic counter method. 2) Viable count / indirect method: a) Plate count technique. b) Membrane filter count. B) DETERMINATION OF CELL MASS: 1) Direct method: a) Dry weight measurement. b) Measurement of cell nitrogen. 2) Indirect method: a) Turbidimetric method. C) DETERMINATION OF CELL ACTIVITY: 1) Measurement of biochemical activity (indirect method). COUNTING OF BACTERIA -TOTAL AND VIABLE COUNTING TECHNIQUES TOTAL COUNTING TECHNIQUE DIRECT METHOD VIABLE COUNTING TECHNIQUE INDIRECT METHOD Also called Also called
PHARMD GURU Page 2 A)DETERMINATION OF CELL NUMBER: 1) TOTAL COUNT (OR) DIRECT METHODS: Total count of micro-organisms in any given suspension indicates the total number of cells which includes both living and dead. The following methods are used for the determination of total count. BREED METHOD: This method is also called as 'Direct microscopic count'. A known volume of cell suspension (0.01 ml) is spread uniformly over a glass slide within a specific area (1 sq. cm). The smear is then fixed, stained, examined under the oil immersion lens and the cells counted. It is impossible to examine entire area (1 sq. cm), therefore practically only a few microscopic areas are observed. Several microscopic fields are counted and an average is taken. The total cells per square cm is then calculated by determining the number of microscopic fields per square cm. COUNTING CHAMBER METHOD: Bacteria can be counted easily and accurately with the Petroff - Hausser counting chamber or Haemocytometer. Counting of microorganisms can be made rapidly and simply with a minimum equipments. In the haemocytometer or counting chamber method, a minute drop of the culture is placed in a tiny, shallow, rectangular glass slide called Neubar's slide. This is a special slide accurately ruled into squares that are 1/400 mm2 in area. A suspension of unstained bacteria can be counted in the chamber, using a phase contrast microscope.
PHARMD GURU Page 3 PROPORTIONAL COUNT METHOD: A standard suspension of particles (plastic beads, number of particles/volume is known) is mixed with an equal amount of cell suspension. This mixed suspension is spread on the slide, fixed and stained. The particles and cells in the microscopic field are counted. An average count of the particles and the cell is taken from the number of fields. Ex: Suppose an average count of 10 particles and 50 cells per field is obtained. If the number of particles in 1 ml of standard suspension is 25,000. Then the number of cells/ml of suspension is:
PHARMD GURU Page 4 ELECTRONIC COUNTER METHOD: An electronic instrument, Coulter counter can be used for the direct enumeration of cells in a suspension. In this technique, the bacterial suspension is passed through a capillary tube. The diameter of this tube is so microscopic that it allows only one cell to pass at a time. The instrument can count thousands of cells in a few seconds. But this system has a disadvantage, in that the Coulter counter counts even dust particles. Hence, the suspension must be absolutely free of any foreign particles. Direct counting methods are rapid and simple. The morphology of cells can also be observed when they are counted under the microscope. The major disadvantage of this method is that it gives the total cell count which includes both viable and non-viable cells. Accuracy also declines with very dense and very dilute suspensions because of clumping and statistical errors, respectively. 1) VIABLE COUNT/INDIRECT METHOD: The principle behind viable count is that all viable cells or spores, under suitable growth conditions multiply and that each cell or spore forms a colony. The number of colonies therefore is the same as the number of viable cells present in the original sample. PLATE COUNT TECHNIQUE: In this method, a measured amount of diluted bacterial suspension is introduced into a Petri plate, after which the agar medium (liquid form 45oC) is added. (Fig. 4.4). Immediately, mix the agar medium with the inoculum by rotating the plate. After the solidification of medium, the plates are incubated at 37oC for 24 hours in an inverted position. A plate having 30 to 300 colonies is selected for counting the number of microorganisms. The plate count technique has certain disadvantages. If the suspension contains different microbial species, then all of them may not grow on the medium used and under the specified conditions of growth. If the suspension is not homogenous and contains aggregates of cells, the resulting colony count will be lower than the actual number of microorganisms because aggregate cells produce a single colony.