Dilution calculations

1. You are interested in determining the number of bacteria in saliva. You spit into a tube, and then do four 1/10 dilution's. From each dilution tube you plate 200 ul onto an appropriate medium, and observe 157 countable colonies on the agar surface after overnight growth. How many bacteria are present in the original sample?

2. A friend of yours tells you that there should be no bacteria in hamburger meat, and having had micro you say not true. To show him/her you do the following: You take 1 gram of meat and blend it in 100 ml of sterile water. You then do the following dilution: 1/10, followed by 1/10 followed by 1/100. You then take 0.1ml from the last dilution, and plate onto an appropriate medium, and find that after 18 hours of growth that there are 125 colonies on the plate. How many bacteria were present in the original sample, per ml of blended material and per gram of hamburger meat?

3. You take a sample from your bacterial culture at 3h post inoculation. You do the following dilutions: ¼, followed by 1/6, followed by 1/8 and then you place 169ul. After 24h you observe 125 colonies. How many CFU/ml did you have in your sample at 3h post inoculation?

1. You are interested in determining the number of bacteria in saliva. You spit into a tube, and then do four 1/10 dilution's. From each dilution tube you plate 200 ul onto an appropriate medium, and observe 157 countable colonies on the agar surface after overnight growth. How many bacteria are present in the original sample

10-1 10-2 10-3 10-4

1ml transferred

9 ml in each test tube

200ul spread on each plate

Plate 1 plate 2 plate 3 plate 4

Colonies 500 350 157 15

CFU/ml= # colonies X DF X 1/volume plated

CFU/ml= 157 X 103 X 1/0.2 = 7.85 X 105

e.g. Hemocytometer calculation

You do a hemocytometer count and find you have 35 cells per primary square. If you only did a 1/100 dilution of your culture sample before you put it on the hemocytometer how many cells/ml do you have in your culture?

Cells/ml= Average # cells in primary square X DF X 10 4

= 35 X 100 X 10 4

= 3.5 X 10 7 cells/ml

Culture media • solutions containing all of the nutrients and

necessary physical growth parameters necessary for microbial growth.

• CHONPS• not all microorganisms can grow in any given

culture medium • many can't grow in any known culture

medium. • In addition to chemical and physical

characteristics, media can be distinguished qualitatively as:

solid vs. broth non-synthetic vs. chemically defined selective Differential, enriched

Solid medium • media containing agar or some

other, mostly inert solidifying agent.

• has physical structure , allows bacteria to grow in physically informative or useful ways

• solid culture media immobilize cells, allowing them to grow and form visible, isolated masses called colonies

• is usually used as: slants , stabs , petri dishes

Broth medium • media lacking a solidifying matrix

Different kinds of media are used:1. Non-synthetic [chemically undefined] medium , Chemically

undefined ingredient: • contains at least one component that is neither purified nor

completely characterized nor even completely consistent from batch to batch.

• May be broth or solid.

2. Chemically defined [synthetic] medium • prepared from purified ingredients and therefore whose exact

composition is known. • Not trivial for fastidious organisms:

• must know exactly what your microorganism's growth requirements are.

• If not known it must be discovered through trial and error and therefore is not a trivial process for fastideous microorganisms.

3. Preprepared medium • Media of many types can be obtained premixed, in an often

dehydrated and powdered state.

Sterilization• Media must be sterilized before use, or cultures will

always have contaminants • Autoclaving- heating the medium to a temperature of

121°C, under 110 kPa (~ one atmosphere) of steam pressure above ambient.

• Filtration- some media components can not be sterilized by heating , can filter through small pore filters

• Dry, or wet and volume are all issues considered when determining how long to autoclave for.

Aseptic technique- ensuring you only grow the organism you want to grow!

• Successful cultivation and maintenance of pure cultures of microorganisms can be done only if aseptic technique is practiced to prevent contamination by other microorganisms.