Enumeration techniques for microbes

1. ENUMERATION
TECHNIQUES FOR
MICROORGANISMS
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2. INTRODUCTION
• Enumeration technique of microorganisms is a process to determine the rate of
microbial growth and death.
• To determine the number of microorganisms present in a sample.
• For example to ability to determine the safety of foods and drugs defends on
knowing the levels of microorganisms present in those products.
• Microbial enumeration takes place by direct method and indirect method
• Direct method : DMC,SPC,Membrane filter,MPN
• Indirect method : turbidity test , metabolic activity and dry weight
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3. DIRECT MICROSCOPIC COUNT
• The most obvious way to count microbial numbers is through direct counting of cells.
• Using a counting chamber is easy, inexpensive and relatively quick . It is also give information about the size
and morphology of microorganisms.
• The petroff-hausser counting chamber for example has small etched squares 1/20 of a mm by 1/20mm and
is 1/50 of a mm deep. The volume of one small square therefore is 1/20,000 of a cubic mm or
1/20,000,000 of a cubic centimetre(cc).
• There are 16 small squares in the large double lined square 1/1250000 cc. The normal procedure is to count
the number of bacteria in five large double lined squares and divided by five to get the average number of
bacteria per large square . This number is then multiplied by 1,250,000 since the square holds a volume of
1/1,250,000 cc, to find the total number of organisms per ml in the original sample
• DMC used in enumeration of bacteria in vaccines and cultures
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4. Petroff-hausser counting chamber
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5. STANDARD PLATE COUNT
• Standard plate count also known as viable cell count , viable cell is defined as a cell
which is able to divide and form a population.
• Standard plate count usually done by diluting the original sample .
• Plating aliquots of the dilutions on to a appropriate culture medium.
• Then incubating the plates under proper condition so that colonies are formed.
• After incubation, the colonies are counted and from a knowledge of the dilution
used the original number of viable cells can be calculated.
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6. Procedure :
• Take 6 dilution tubes, each containing 9ml of sterile saline .
• Dilute 1ml of sample by withdrawing 1ml of sample and dispensing this 1ml into first dilution
tube .
• Using the same procedure withdraw 1 ml from the first dilution tube and dispense into the
second dilution tube ,subsequently withdraw 1 ml from second dilution and dispense into the
third dilution tube and continue doing this from tube to tube until the dilution is completed.
• Transfer 1ml from each of The last three dilution tubes onto the surface of the corresponding
agar plates.
• Incubate the agar plates at proper condition for microorganisms to grow colonies.
• Choose a place that appears to have between 30 and 300 colonies.
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7. SPC
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8. MEMBRANE FILTER
• Membrane filters are used to determine microbial numbers from the counts of the
colonies.
• The filter (polycarbonate membrane) have small pores, enough to trap bacteria.
• In this technique , a sample is drawn through special membrane filter and is then
placed on an agar medium or a pad soaked with liquid media and incubated until
each colony forms a separate colony
• A colony count gives the number of microorganisms in the filtered sample and
special media can used to select for specific micro oraganisms
• It is used in enumeration of bacteria in milk, water, food, etc.
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10. MOST PROBABLE NUMBER
• This enumeration based on the statistic and it allows estimating the concentration of viable
microorganisms in a sample by means of replicate liquid broth growth in serial dilution.
• It is commonly used in estimating microbial population in soil, water and agricultural products.
• MPN is most commonly applied for quality testing of water
• Water to be tested is diluted serially and inoculated in lactose broth
• Coliforms if present in water utilize lactose present in the medium to produce acid and gas.
• The presence of acid is indicated by colour change of the medium and the presence of gas is
detected as gas bubbles collected in the inverted Durham tube present in the medium.
• The number of total coliforms is determined by counting the number of tubes giving positive
reaction and comparing the pattern of positive (the number of tubes showing growth at each
dilution) with STANDARD STATICAL TABLE.
• MPN TEST IS PERFORMED IN 3 STEPS : Presumptive test , confirmatory test and completed
test
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11. Presumptive test
• It is a screening test to sample water for the presence of coliform organisms
• If the presumptive test is negative ,no further testing is performed and the
water source is considered microbiologically safe.
• If however, any tube with series shows acid and gas ,the water is considered
unsafe and confirmed test is performed on the displaying a positive reaction.
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14. Confirmed test
• Some microorganisms other than coliforms also produce acid and gas from lactose fermentation. In order to
confirm the presence of coliform ,confirmatory test done.
• From each test fermentation tubes with positive results transfer one loopfull of medium to 3 ml lactose-
broth or brilliant green lactose fermentation tube ,to an agar slant
• Incubated and identify the organisms by gram staining
Completed test
. since some of the positive results from the confirmatory test may be false ,it is desirable to do completed
tests, for this inoculum from each positive tube of the confirmatory test is streaked on a plate of EMB agar
. In this test we use selective media
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15. TURBIDITY TEST
• A spectrophotometer or calorimeter can be used for turbidometric measurements of cell mass.
• A spectrophotometer is used to determine turbidity ("cloudiness") by measuring the amount of light that passed through a suspension of cells.
• More cells = more turbidity; more turbidity = less light passing through the suspension
• However, the culture to be measured must be dense enough to register s Moreover, it may not be possible to measure cultures grown in deeply
coloured media or cultures that contain suspended material other than bacteria.
• It must also be recognised that dead as well as living cells contribute to turbidity.
• (%T) percent transmission - fewer cells present (less turbidity) and thus will allow more light to pass through.
• Therefore the %T is higher when the cell number is lower. Absorbance is the opposite of %T.
• More light is absorbed when more cells are present and thus absorbance goes up as turbidity (OR cell number) goes up.
It is used for microbial assay , estimation of cell crop in broth, cultures or aqueous suspensions.
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17. METABOLIC ACTIVITY OF MICROBES
• Another indirect way of estimating bacterial numbers is measuring the metabolic
activity of the population (for example, acid production or oxygen consumption,
nutrient utilization, waste production, pH, etc).
• The assumption is that the amount of acid produced or oxygen consumed under
specific conditions and during a fixed period of time is proportional to the
magnitude of bacterial population.
• Admittedly, the measurement of acid or any other end product is a very indirect
approach to the measurement of growth and is applicable only in special
circumstances.
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18. Metabolic activity
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19. DRY WEIGHT
• This is the most direct approach for quantitative measurement of a mass of cells
• It can be used only with very dense suspensions and the cells must be washed free of
all extraneous matter.
• Dry weight may not always be indicative of the amount of living material in cells.
• The cells growing in liquid medium are collected by centrifugation washed dried in
an oven and weighed
• This technique may also used for measuring the growth of fungi,
• It is time consuming and not very sensitive, because bacteria weigh is so little it is
necessary to centrifuge several hundred millilitres of culture to collect a sufficient
quantity.
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20. REFERENCES
• Microbiology
-Michael J. Pelczar,JR
-E.C.S.Chan
. DAIRY MICROBIOLOGY
-Parihar and Parihar
.https://www.sas.upenn.edu/LabManuals/biol275/Table_of_Contents_files/14-Enumeration.pdf
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22. THANK YOU
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