Culture Media and Reagent Calculations

A. Essential components of a culture media

1. Nitrogen sources
2. Carbon sources
3. Energy sources
4. Minerals
5. Growth factors

B. Optional substances to be added

1. Buffer salts
2. Indicator dye
3. Reducing substances
4. Selective agents

C. Culture media may also be categorized as:

1. Dehydrated- all components are pre-mixed in a fixed formulation
2. Compounded- each component has to be prepared individually

D. Can also be categorized based on the amount of solidifying agent (usually agar)
1. Solid (1.5 – 2% agar)
2. Semi-solid (0.6 – 0.8% agar)
3. Liquid/broth (no agar)

Media Preparation/Reagent Calculations

1. Ratio and Proportion

Volume of media to be dispensed on glassware

Petri dish/ Plates 15-20ml
Slants 6-8ml/big test tube
3-5ml/small test tube
Stabs/Broth 10ml/ big test tube
5ml/small test tube

Example: Prepare Nutrient Agar for 15 slants (big tubes) and 20 plates

Formulation of Nutrient Agar

Component Amount
Beef extract 3 grams
peptone 0.5% w/v
agar 17g
Distilled water 1000ml

2. Dilutions/Concentration

a. One-step dilution
Dilution = volume transferred/total volume
Dilution factor= 1/dilution

Example 1: A 1.0 g sample of dextrose monohydrate was transferred to 99ml of distilled water, what is the resulting dilution? What is
the resulting dilution factor?

Example 2: 20.0ml of sodium lactate was dissolved in 500ml of de-ionized water, what is the resultant dilution? What is the resulting
dilution factor?

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 b. Serial dilution
Formulae:
Dilution = (volume transferred/total volume) previous dilution
DF = Initial concentration/ final concentration

Example3: 20 grams of a drug sample was diluted with 180ml of phosphate buffer (as diluent). This solution was labeled as the first
dilution. From this solution, an aliquot of 1ml was obtained and was further diluted with 9ml of phosphate buffer this was labeled as
nd
the 2 dilution. From this solution another 5ml aliquot was taken and was diluted with 35ml of phosphate buffer. This was labeled as
rd
the 3 dilution.

a. Illustrate a dilution scheme for the procedure mentioned above.

b. Calculate the resultant dilution of the first, second and third dilutions.

c. Calculate the resultant dilution factors of the first second and third dilutions.

C. Concentration adjustment

C1V1 = C2V2
C1V1/Va=C2V2/Vb

C1 = initial concentration
C2 = final concentration
V1 = initial volume
V2 = final volume
Va = Initial volume
Vb = Final volume

Example 4: 20ml of 0.2%(w/v) iodine tincture is to be prepared from a stock solution of 10%w/v iodine tincture. How much stock
solution should be diluted with distilled water to come up with the desired concentration?

Example 5. How many times should a polymyxin B sulfate solution be diluted with phosphate buffer to reduce its concentration from
20%w/v down to 2.0x10^-6%w/v?

Construct a dilution scheme for the problem on example 5 and label each tube with the appropriate dilution level.

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Example 6 In a given formulation, 2ml of 2%w/v crystal violet should be added per 50ml total volume of medium ABC. You only
have a stock solution of 10% w/v crystal violet, how much of this stock solution should be added when preparing 30ml of mediu m
ABC?

3. Common expressions of concentration [percent; ppt; pph; (%w/w); (%w/v); (%v/v)]

The metric system comprises three basic units of measurement:

distance measured in meters, volume measured in liters, and mass measured in grams. In order to designate larger and
smaller measures, a system of prefixes based on multiples of ten is used in conjunction with the basic unit of
measurement. The most common prefixes are

Prefix
kilo 10^-3 1000
centi 10^-2 1/100
milli 10^-3 1/1,000
micro 10^-6 1/1,000,000
nano 10^-9 1/1,000,000,000
pico 10^-12 1/1,000,000,000,000

Concentration w/w w/v v/v

Parts per hundred (pph) or % g/100g g/100ml ml/100ml
Parts per thousand (ppth) g/kg g/liter ml/liter
Parts per million (ppm) mg/kg mg/liter µl/liter
Parts per billion (ppb) µg/kg µg/liter nL/liter
Parts per trillion (ppt) ng/kg ng/liter pL/liter

Example 7: You need to prepare 350ml of 0.85% (w/v) NaCl solution, how much NaCl do you need to weigh?

Example 8: You need to prepare 50ml of 20ppm of lidocaine hydrochloride. How many grams do you need to prepare?

4. Molarity (M)

Molarity = moles/ liter or grams/molecular weight/liter (g/MW/L)

Example 9: How much KCl do you need to weigh if you need to prepare 200ml of 0.2M of KCl?

5. Normality (N)

Normality = eQ/L; (number of equivalents of solute per liter of solution)

eQ = g/Empirical Weight

Empirical weight = MW/n n= number of reacting units/ no. of H+ or OH- ions/ no. of protons exchanged in a
neutralization reaction/ no. of electrons per mole in a redox half-reaction.

Normality = nM

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 Example 10: How much NaOH pellets do you have to weigh to prepare 500ml of 0.2N of NaOH solution?

Example 11: Prepare 300ml of 0.1M H 2SO4 from a stock solution of 5N H2SO4. Compute the needed amount and describe
the manner of preparation and dilution.

6. Computing equivalent mass of hydrous from anhydrous compounds and vice versa.

Wt. of hydrous form = wt of anhydrous form x (MW hydrous/MW anhydrous )

Example12: The formulation says you have to add 1.5g magnesium sulfate (anhydrous), MgSO 4 per liter of distilled water
however, only magnesium sulfate pentahydrate is available in the laboratory. How much magnesium sulfate pentahydrate
should you weigh to get the equivalent mass of the anhydrous form?

Example 13: The formulation says you need 2 grams of ferrous chloride monohydrate (FeCl 2) per liter of medium XYZ.
You are to prepare 50ml only of medium XYZ. In the laboratory however only ferrous chloride tetrahydrate is available in
the laboratory, how much of this reagent do you need to weigh in order to get the equivalent amount you need for 50ml of
medium XYZ?

Combined problem:

Prepare Nutritious medium enough for the following:

5 plates (standard)
3 big stabs
3 big slants
5 small stabs
5 small slants
1 250ml Erlenmeyer flask
2 125ml Erlenmeyerflask

Formulation of Nutritious medium per 500ml of medium

glucose 1.0 g (available as a 15%w/v stock solution)

maltose 0.1%w/v
soy peptone 2.0 g
Casein hydrolysate 5000 mg
Methylene blue 2ml of 0.1%w/v stock solution
Ammonium sulfate 5ml of 1% stock solution (available as 5% stock solution)
Lead acetate 0.005%w/v (available as 1% stock solution)
agar 2% w/v
Distilled water ?

Describe the manner of preparation from weighing and combining the computed amounts of the individual medium
components to sterilization.

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