Lesson 1 Activity In this experiment you will test milk for total number of viable microorganisms by the Standard Plate Count assay.

Experiment

In this experiment you will test milk for total number of viable microorganisms by the Standard Plate Count assay. This involves spreading a specific amount of a diluted sample onto a Standard Methods Agar petri plate, incubating the plate for 48 hours, and then counting the number of colonies on each plate and calculating the number of microorganisms per ml in the original sample. As QA inspectors, you want to determine if the milk sample meets the specification for Grade A milk.

Materials

  1. Grade A pasteurized milk
  2. Alternate: Temperature-abused pasteurized milk

Equipment and Supplies

  1. 2, 99 ml serial dilution bottles
  2. 1. l ml sterile pipet (three required)
  3. Pipet-aid or pipet bulb
  4. 4 sterile inoculating loops
  5. 4 pre-poured, sterile Standard Methods Agarplates
  6. Felt-tip, black, marking pen
  7. Magnifying glass
  8. Microbiological incubator, 35C

Procedure

Today

  1. Wash your hands and sanitize a work bench. Obtain a milk sample and all other supplies necessary to begin.
  2. Label your dilution bottles 10[-2] and 10[-4], respectively. Label the bottom of your plates with your name or initials, sample being tested, and the dilution factor: 10[-2], 10[-3], 10[-4], and 10[-5], respectively.
  3. Attach the pipet bulb to a sterile 1.1 ml pipet and carefully, without contamination, transfer 1.0 ml from the milk carton to the first dilution bottle, labelled 10[-2]. This represents 1.0 ml in a total of 100 ml, or a 1/100 dilution which equals 10[-2]. Hold onto the pipet so as to not get it contaminated, cap the bottle and shake quickly 25 times in a one-foot arch.
  4. Uncap the bottle and the 10[-4] bottle. Using the same pipet, transfer 1.0 ml from the 10[-2]bottle to the 10[-4] bottle. Repeat the shaking procedure for this second bottle. This bottle represents a 1/10,000 dilution which equals 10[-4]. Discard the pipet.
  5. Uncap the bottle (if there was more than a minute delay after shaking, then re-shake the bottle first) and insert a new sterile 1.1 ml pipet, with pipet bulb attached, into it. Draw out 1.1 ml. To the 10[-5] plate, deposit 0.1 ml of the sample and to the 10[-4] plate, deposit 1.0 ml of the sample. Spread each sample thoroughly on the surface of each plate using a sterile inoculation loop.
  6. Re-shake the 10[-2] bottle, uncap it, and insert a clean 1.l ml pipet with a pipet bulb attached. Draw out 1.1 ml. To the 10[-3] plate deposit 0.1 ml and to the 10[-2] plate deposit the remaining 1.0 ml. Spread each sample using a sterile inoculating loop as done previously.
  7. Invert the petri plates and tape together. Place in an incubator set at 35C for 48 hours.

48 Hours Later

  1. After 48 hours of incubation, remove the petri plates. Be careful not to turn the plates right-side up if there is excessive condensation on the cover plate. Count all colonies using your marking pen and magnifying glass. Record the total number of colonies for each plate on a worksheet (below).
  2. Calculate the number of microorganisms per milliliter of milk sample by multiplying the total number of colonies for a particular plate times the inverse of the plate's dilution factor. Record your answer on a worksheet (see below).

Record Your Answers

 

Sample data set

Dilution: 10[-2] 10[-3} 10[-4] 10[-5]

Count: TNTC[a] 728[b] 85[c] 9[d]

Calculation: 85 x 10[+4]

85 x 10,111 = 850,000 microbes per ml

This milk would most likely be sour or bad.

Notes

  • a - TNTC - Too Numerous To Count. There's just too many.
  • b - More than the acceptable range of 20 to 200 colonies per plate.
  • c - Good count. Between 20 and 200 colonies per plate.
  • d - Not an acceptable count; out of the acceptable count range.

Your Data Set

Dilution: 10[-2] 10[-3] 10[-4] 10[-5]

Count:_____________________________________________________

Calculation:_________________________________________________

Your answer: ________________________________ microbes per ml