Beruflich Dokumente
Kultur Dokumente
Enumeration
S. Y. B. Pharm Sem IV
2017-18
Ms. Heena Bhojwani
Bacterial Reproduction
• Can reproduce sexually and asexually
Methods for reproduction
1. Binary Fission
2. Budding
3. Fragmentation
4. Formation of conidiospores and
sporangiospores
Binary Fission
• Asexual process
• Results in division
of a cell into two
vegetative cells
• Also called as
transverse binary
fission
• Examples:
• Bacillus subtilis
• Streptococcus fecalis
Budding
• Dilution is important:
colonies will have to be counted and with a concentrated sample
there may be too many colonies than can be accurately counted
• Plating is important:
count of only the living cells is required in this procedure (only
living cells will be able to multiply and form colonies)
Important considerations
• Only living cells are counted
• Keep in mind that if the organism normally forms
multiple cell arrangements, such as chains, the colony-
forming unit may consist of a chain of bacteria rather
than a single bacterium
• Some of the bacteria may be clumped together.
• Therefore, when doing the plate count technique, we
generally say we are determining the number of Colony-
Forming Units (CFUs) in that known dilution
• By extrapolation, this number can in turn be used to
calculate the number of CFUs in the original sample
• Bacterial counts by these methods are usually expressed
as colony forming units per milliliter (CFU/mL)
• Normally, the bacterial sample is diluted by factors of 10
and plated on agar.
• After incubation, the number of colonies on a dilution
plate showing between 30 and 300 colonies is
determined.
• A plate having 30-300 colonies is chosen because this
range is considered statistically significant
Procedure
1. Using sterile technique, transfer 1 mL sample to the first dilution blank.
Mix the bottle by inverting it 20 times. Label the bottle "10-1."
2. Using a fresh pipette, transfer 1 mL from the first blank to the second
blank. Mix as before. Label the second bottle "10-2."
3. Using a fresh pipette, transfer 1 mL from the first blank to the second
blank. Mix as before. Label the second bottle "10-2“.
4. Using a fresh pipette, transfer 1 mL from the first blank to the third blank.
Mix as before. Label the second bottle "10-3“.
5. Using a fresh pipette, transfer 1 mL from the first blank to the forth blank.
Mix as before. Label the second bottle "10-4“.
6. Using a fresh pipette, transfer 1 mL from the first blank to the second
blank. Mix as before. Label the fifth bottle "10-5“.
7. Label the Petri dishes: 10-2, 10-3, 10-4, 10-5, and 10-6, respectively.
8. Transfer liquid from the dilution blanks to the Petri dishes. Use a separate
pipette for each blank, not for each plate (i.e. if more than one plate uses
liquid from a single blank, a single pipette may be used for that blank).
9) One at a time, add a tube of molten nutrient agar
to each Petri dish. After adding the agar, gently
swirl the dishes in pattern for 30 seconds to mix
the bacteria with the agar.
10)After the agar has thoroughly solidified, incubate
the plates at 37°C for 24 to 48 hours.
11)Count the number of colonies on a plate that has
between 30 and 200 colonies.
12)Any plate which has more than 200 colonies is
designated as "too numerous to count" (TNTC).
13) Plates with fewer than 30 colonies do not have
enough individuals to be statistically acceptable
• Calculate the number of bacteria (CFU) per
milliliter or gram of sample by dividing the
number of colonies by the dilution factor
multiplied by the amount of specimen added to
agar plate.
• To compute the number of CFU/mL, use the
formula:
c = concentration, CFU/mL
n = number of colonies
d = dilution blank factor
s = volume transferred to plate.
Example for calculation
• You count 46 (n) colonies on your plate
• You put 1 ml of bacterial culture into 99 ml
of saline and plated 0.1 ml (s)
• Dilution 1/100 (d)
• CFU = 46/ 10-2 * 0.1
= 46*100*10
=46000 CFU/ml
Membrane Filter Technique
1. Collect the sample and make any necessary
dilutions.
2. Select the appropriate nutrient or culture
medium. Dispense the broth into a sterile
Petri dish, evenly saturating the absorbent pad.
3. Flame the forceps, and remove the
membrane from the sterile package.
4. Place the membrane filter into the funnel
assembly.
5. Flame the pouring lip of the sample container and pour the sample
into the funnel.
6. Turn on the vacuum and allow the sample to draw completely
through the filter.
7. Rinse funnel with sterile buffered water. Turn on vacuum and allow
the liquid to draw completely through the filter.
8. Flame the forceps and remove the membrane filter from the
funnel.
9. Place the membrane filter into the prepared Petri dish.
10. Incubate at the proper temperature and for the appropriate time
period.
11. Count the colonies under 10 - 15 X magnification.
12. Confirm the colonies and report the results.
Commonly used Filters
Advantages of Membrane Filter
Method
• Permits testing of large sample volumes.
• Reduces preparation time as compared to many traditional
methods.
• Allows isolation and enumeration of discrete colonies of
bacteria.
• Provides presence or absence information within 24 hours.
• Effective and acceptable technique. Used to monitor drinking
water in government laboratories.
• Useful for bacterial monitoring in the pharmaceutical,
cosmetics, electronics, and food and beverage
• industries.
• Allows for removal of bacteriostatic or cidal agents that would
not be removed in Pour Plate, Spread
• Plate, or MPN techniques.
Turbidimetric Measurement