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8.

Enumeration of bacteria
Viable count
1. pour plate: original sample is diluted several
times to thin out the population. The most
diluted samples are then mixed with warm agar
and poured into petri dishes. After agar has
hardened, each cell is fixed in place and forms
an individual colony. Total # of colonies equals
the # of viable microorganism in the diluted
sample
2. spread plate: a small volume of diluted microbial
mixture is transferred to the centre of an agar
plate and spread evenly over the surface with a
sterile bent-glass rod. The dispersed cells
develop into isolated colonies.
3. membrane filter : used when expected count is
below ~ 20cfucm-3 ( colony forming units). Have
pores small enough to trap bacteria. Filter then
placed on agar medium/pad soaked with liquid
media and incubated until each cell forms a
separate colony.
4. drop plate method (Miles and Misra) : Suspension is
diluted to 20-80 organisms in 0.02 ml. Using a dropping
pipette set at a height of 10 mm from the agar surface,
each dilution is dropped on to each plate at a rate of one
drop a second, i.e. 6 to 8 drops/dish.
5. most probable number (MPN) : an older method of
counting devised before the introduction of solid media.
Based on statistical sampling theory, to calculate the
probability of obtaining # viable organisms in a sample of
stated size when the count is known. Will give a pattern of
growth and no growth which the count may be calculated.
Errors are large.
total count
1. (i)counting chamber/haemocytometer/electronic cell
counters: direct counting on designed slide ( Petroff
Haussen). Quick way to estimate microbial cell #.
(ii)A shallow well of known volume is indented into the
surface of a microscope slide inscribed with squares of
known area (depth & area).

# of bacteria/# of squares * 25 squares * area * depth = # of bacteria/mm

2. Breed method : developed for milk A known volume of


material is spread evenly over a known area of slide, fixed
and stained and the mean # of cells in each field is
counted and averaged.. Knowing the magnification, the
original count can be determined.