Experiment 8: Standard Plate Count

what is a direct count?

gives total # of bacteria, including dead and viable cells

what is the standard plate count?

only gives viable cell count using colonies formed on plates after incubation

what is SPC used for?

determining number of organisms in water, milk, and food

what does a typical plate count procedure include?

serial dilutions and pour plate method to detct colonies on a growth medium

why do we dilute the starting material into dilution tubes?

bc the SM has unknown amt of bact, so we dilute w known aliquot of diluent to reduce microbial #s to quantifiable range

what does a tube of 9mL diluent and 1mL culture mean?

10^-2 dilution factor

culture represents 1/100 of contents in TT

dilute until when?

statistically countable range (25-250 colonies)

what is produced to after pouring and solidifying plates?

viable cells to produce visible colonies

each viable cell will make a colony

as dilutions increase?

#. of colonies decrease

what is CFU and why do we use it?

Colony forming units

bc clump of cells (not js one) will also make js one colony and its hard to break these clumps apart so we use CFUs

#. of colonies counted x dilution factor

= #. of bacteria/colonies per mL

or #. of CFU per mL

Experiment 8 learning objectives

1. perform transfer for serial dilutions

2. demonsrate aseptic technique

3. understand CFUs and DF and calculate

Experiment 8 Material

1. E.coli (24-48hrs)

2. 4 9.9 blank TT w sterile water

3. 5 sterile petris

4. P1000 w blue tips

5. 120 mL molten nutrient agar in 50 deg water bath

Exp 5 method (14)

1. label tubes 10^-2 , 10^-4, 10^-6 , 10^-8 TT and 10^-5 , 10^-6 , 10^-7 , 10^-8 , 10^{-9 petris}

2. transfer 0.1mL (100uL) of culture to 10-2 tube

3. transfer 0.1mL of 10-2 into 10-4

4. transfer 0.1mL of 10-4 to 10-6

5. transfer 0.1mL of 10-6 to 10-8

6. strat at highest dilution 10-8, transfer 0.1mL to 10-9

7. transfer 1mL of 10-8 to 10-8

8. transfer 0.1mL of 10-6 to 10-7

9. transfer 1mL of 10-6 to 10-6

10. transfer 0.1mL of 10-4 to 10-5

11. get agar from water bath, pour 15mL into each plate to cover ¾ of plate

12. rinse agar bottle and drain in sink, if solid = garbage

13. figure 8 pattern each plate to mix, rotate plate 45 deg and swirl, dont splash on lid.

14. solifify for 10-15 mins. invert and incubate at 37 deg for 48hrs

if 0.1mL of 10-6 plate has 56 colonies, how many cells per mL in original culture?

56/0.1 = 560 × 10⁶ = 5.6 × 10⁸ cells/mL

Results - using Quebec colony counter

1. dont remove lid and put on counter so the bottom of plate is closest to you

2. start at top using grid lines and use mechanical hand tally count every colony small or big

3. calculate # org/mL by multiplying # by DF only use two sig figs

CFU/mL formula

Results - Plate vs # of colonies

10^-5 = too many

10^-6 = 94

10^-7 = 41

10^-8 = 32

10^-9 = 0