4.3.1 direct methods

what are the different types of direct methods?

direct microscopic count

plate count methods

membrane filtration

most probable number (MPN)

what is direct microscopic count?

this method involves counting the number of bacterial present in a measured volume of diluted culture

what makes up the total count for direct microscopic count?

both viable and dead cells are counted

what is used to perform direct microscopic count?

special counting chamber called the Petroff-Hauser counting chamber

what does the Petroff-Hauser counting chamber do?

count the number of cells present in specific volume of culture, then finally expressed as cells/ml

what are the advantages of direct microscopic count in terms of results?

rapid with immediate results, no incubation required

what are the disadvantages of direct microscopic count in terms of counting?

difficult to count if cells are motile or clumped and insensitive which required at least 10 power 6 cells/ml

what are the disadvantages of direct microscopic count in terms of humans

tedious, tiring and large errors are likely

what is the plate count method?

makes a viable count, only living cells will be counted?

how to preform plate count method in terms of preparation of sample?

begin with serial dilutions of the sample

how to perform plate count method in terms of pouring?

plate out desired dilutions in duplicates

how to perform plate count method in terms of counting>

count only plates with 25 - 250 cfu

what does cfu mean?

colony forming units

how to calculate cell density (cfu/ml)

average cfu xx dilution factor / volume of inoculum

advantages of plate count method in terms of sensitivity

reasonable sensitivity and does not require complicated equipment

what is the purpose of plate count method?

it is often used for environmental and food samples

disadvantages of plate count method in terms of waiting

slow and tedious, requires overnight incubation

disadvantages of plate count method in terms of accuracy

accuracy depends on counting large numbers of cfu

what is membrane filtration?

used to concentrate the bacteria for counting

where is the bacteria taken from for membrane filtration?

water samples are very small taken from lakes or reservoirs

what is the 1st step of membrane filtration? (hint: what does the sample go through?)

large volume of sample is filtered through a 0.45 membrane filter

what is then 2nd step of membrane filtration? (hint: what happens after the bacteria is trapped?)

bacteria is trapped at the membrane filter and the membrane filter is separate from the filtration unit

what is the 3rd step of membrane filtration? (hint: what happens after the filter is separated?)

the membrane filter with bacteria is placed on the agar and bacterial cells are grown on the membrane filter and colonies will be formed after incubation

what is the application for membrane filtration?

used when the samples have low cell densities

what are the advantages of membrane filtration?

large volumes of samples can be processed and good for samples with low cell densities

what is the disadvantage of membrane filtration?

tedious and expensive

what is MPN?

a statistical approach, the more cells in a sample, the more dilutions are needed to reduce the numbers to zero

what is the 1st step for MPN? (hint: preparation)

make 3 consecutive serial dilutions of the sample

what is the 2nd step for MPN? (hint: inoculation)

for each of these dilutions. inoculate into 3 or 5 tubes of suitable growth medium

what is the 3rd step of MPN? (hint: incubation)

incubate and score the number of positive tubes for each solution

what is the purpose of MPN?

used for environmental, food and water samples also good for samples with low cell densities

what is the disadvantages of MPN?

only provide an estimated range of microbial numbers

what are the advantages of MPN?

often used for the enumeration of coliforms and E. coli using combination of specific media and incubation conditions