1.2.2 Preparation of Culture media and Quality Control

Question 1

What is the first thing to do?

Answer 1

Check the manufacturer instruction on how to prepare (Since every manufacturer have different Volume or total volume in preparation)

Question 2

How to prepare MAC?

Answer 2

1. prepare 50g / 1000mL of distilled water
2. Weigh the 50g MAC on a foil then put it on Erlenmeyer flask

Question 2

How to prepare MAC?

Answer 2

1. prepare 50g / 1000mL of distilled water
2. Weigh the 50g MAC on a foil then put it on Erlenmeyer flask
3. Then add the half of the total volume (500 mL) of distilled water and heat to boil to dissolve the MAC (Magnetic stirrer)
4. Add the remaining volume to the flask (500mL) and cover it with Foil
5. Sterilize it with Autoclave (121 C for 15 min, 15 psi)
6. Allow to cool at 45-50 C in a water bath
7. Poor approx 20-25 mL (18-20mL) in a 90 mL petri dish and allow to solidify for 15-20 mins
8. Store at 2-8 C in an inverted position until further use

Question 3

Why Water bath is used to cool down the media?

Answer 3

It controls the cooldown temperature of the media and ensure the correct range of temp when dispense

Question 4

Safest temp in autoclave and cooldown

Answer 4

121 C, 15mins, 15 psi
45 - 50 C

Question 5

How to prepare trypticase soy broth?

Answer 5

1. Prepare 29.77 g / 1000mL of distilled water
2. Weigh the TSB at 29.77 then put it on a beaker
3. Heat to boil at half volume until it dissolves (Magnetic stirrer)
4. Add the remaining volume to the beaker
5. Dispense it in a test tube (Depends on Vol, and orientation)
6. Autoclave it (121 C, 15mins, 15 psi)
7. Allow to solidify for 15-20 mins (Agar, not broth)
8. Store at 2-8 C until further use

Question 6

What happens when agar does not dissolve properly?

Answer 6

Uneven distribution of solidification

Question 7

What is the reason why Dispense first the TSI in the tube before autoclaving?

Answer 7

Prevents contamination

Question 8

Why you should not tighten the cap of TSI tube during autoclave?

Answer 8

To allow the steam to enter in the tube for proper sterilization

Question 9

Prep: 36g of dehydrated EMBA/1000mL of water
Question: How many grams of EMB agar is needed to prepare a volume of 750 milliliters?

Answer 9

36g x 750 mL = Answer/1000mL
= 27g of dehydrated EMBA

Question 10

Prep: 29.77g of dehydrated TSB 1000mL of distilled water

Question: How many grams of TSB is needed to prepare a volume of 600 milliliters

Question: How many grams of Bacteriological agar is needed to prepare 1.5% TSI?

Answer 10

29.77 x 600mL = Answer / 1000mL
= 17.86g of dehydrated TSB

1.5%/100 = 0.015 x 600mL = 9 grams

(TSB first then agar then water)

Question 11

NOTE:

Answer 11

The total volume depends on how much u need (Ex. 3mL/tube and u need 300 tubes so you need 300 mL total volume)

Question 12

Quality Control

Answer 12

• All media must be tested before use
• Each medium must be tested with organisms expected to grow or give positive reaction as well as with organisms expected not to grow or to produce a negative reaction
• The medium should be tested for tested for sterility and pH
• The organisms selected for QC should represent the most fastidious organisms for which the medium was designed.
• QC testing should be performed according to CLSI recommendations
• Expiration dates must be established

Question 13

How to adjust the pH on a media?

Answer 13

1 molar sodium hydroxide (Base)
1 molar hydrochloric acid (Acid)

Question 14

What are he information for QC forms?

Answer 14

Amount prepared
Source of each ingredient
Lot number
Sterilization method
Preparation and Expiration date
Name of the preparer

Question 15

The start of 15 minutes in Autoclave happens in what Temp?

Answer 15

121 C

Question 16

How many mL should dispence in 90mm plate?

Answer 16

18-20 mL?

Question 17

18-20 mL / 90 mm plate said by?

Answer 17

ISO 11133

Question 18

If ingredients that may destroy during autoclave, what is there method for sterilization?

Answer 18

Membrane filtration (0.2 - 0.45 micrometers in diameter)