Experiment Title: Preparing and Sterilizing Media

Calculations:

Table 1: TABLE SHOWING THE AMOUNT OF WATER AND MEDIA MIX REQUIRED TO MAKE THE CORRECT AMOUNT OF MEDIUM

Bottle Label (g/L)

Media Mix(g)

Water(ml)

Agar %(%)

Agar(g)

Final volume

42

21.0

500

--

--

500

50

25.0

500

--

--

500

40

10.0

250

--

--

250

50

22.5

450

--

--

450

23

13.8

600

1.5

9

600

8

0.8

100

2

2

100

24

36.0

1500

1

15

1500

42

14.7

350

0.8

2.8

350

8

13.2

1650

1

16.5

1650

33

74.25

2250

2

45

2250

Bottle Label:

1. Bottle label = media mix ÷ water (liters)

Bottle label = 25 g ÷ 0.5 L

Bottle label = 50 g/L

1. Bottle label = media mix ÷ water (liters)

Bottle label = 0.8 g ÷ 0.1 L

Bottle label = 8 g/L

1. Bottle label = media mix ÷ water (liters)

Bottle label = 13.2 g ÷ 1.65 L

Bottle label = 8 g/L

Media Mix:

1. Media mix = bottle label × water (liters)

Media mix = 42 g/L × 0.5 L

Media mix =  21 g

1. Media mix = bottle label × water (liters)

Media mix = 50 g/L × 0.45 L

Media mix = 22.5 g

1. Media mix = bottle label × water (liters)

Media mix =  23 g/L × 0.6 L

Media mix = 13.8 g

1. Media mix = bottle label × water (liters)

Media mix = 24 g/L × 1.5 L

Media mix = 36 g]

Water:

1. Water (liters) =  media mix ÷ Bottle label

Water (liters) = 10 g ÷ 40 g/L

Water (liters) = 0.25 L

Water (milliliters) = 250 ml

1. Water (liters) =  media mix ÷ Bottle label

Water (liters) = 14.7 g ÷ 42 g/L

Water (liters) = 0.35 L

Water (milliliters) = 350 ml

1. Water (liters) =  media mix ÷ Bottle label

Water (liters) = 74.25 g ÷ 33 g/L

Water (liters) = 2.25 L

Water (milliliters) = 2250 ml

Agar %:

1. Agar % = Agar ÷ Water

Agar % = 2 g ÷ 100 ml

Agar % = 2%

1. Agar % = Agar ÷ Water

Agar % = 16.5 g ÷ 1650 ml

Agar % = 1%

Agar (g):

1. Agar = (Agar % ÷ 100) × Water

Agar = (1.5% ÷ 100) × 600 ml

Agar = 0.015 × 600 ml

Agar = 9 g

1. Agar = (Agar % ÷ 100) × Water

Agar = (1% ÷ 100) × 1500 ml

Agar = 0.01 × 1500 ml

Agar = 15 g

1. Agar = (Agar % ÷ 100) × Water

Agar = (0.8% ÷ 100) × 350 ml

Agar = 0.008 × 350 ml

Agar = 2.8 g

1. Agar = (Agar % ÷ 100) × Water

Agar = (2 ÷ 100) × 2250 ml

Agar = 0.02 × 2250 ml

Agar = 45 g

Final Volume:

The final volume would be the same as the water volume.

Questions:

• What is the temperature in degrees F when it is 170°C? 338°F.

1. Why was it unnecessary to adjust the pH of the nutrient broth?

1. Nutrient broth has already been adjusted by the manufacturer to a specific pH and also contains buffering agents to maintain it, as long as the broth has been made up as specified. Any adjustment to the pH would kill the microorganisms and inhibit their growth.

2. How would you adjust the pH of an agar medium?

1. Calibrate the pH meter with buffer solutions, washing the electrode after each solution. Measure the pH of the agar medium, if it is not at the optimal pH use dilute NaOH to increase the pH of dilute HCl to decrease the pH, adjusting until the correct pH of the media is achieved.

3. Why has it been impossible to grow some types of microorganisms on a defined medium?

1. Defined media, also known as synthetic media are pure media where the concentration and quantity of all the chemicals are known and none of the components is microbial or animal-derived such as bovine serum which is commonly added to culture media as a source of nutrients for the microorganisms. This type of medium may be unfit to grow certain microbes as the components of the media may lack the nutrients such as amino acids, vitamins and nucleotides or the quantities of these nutrients may not be sufficient for growth so energy would be used to synthesize the compounds for growth. Their environmental conditions may also make it impossible as some microbes will need helper microorganisms to produce an environment conducive to growth through nutrient production or by-product removal.

4. What are the advantages of complex media in the general culturing of microorganisms

1. Complex media contain many animal or plant tissue-derived compounds such as yeast extract, peptones from plants or albumin serums whose quantity and concentrations are unknown. It is cheaper to produce than defined media because it is harder and more expensive to manufacture purified forms of the compounds necessary for that microbial growth as well as to obtain in large quantities. This type of media is ideal for growing microbes with nutritional requirements that are unidentified and for rapidly bacterial growth such as heterotrophic organisms. As the media is nutrient-rich, it provides the optimal environment for fastidious bacteria like streptococcus pneumoniae, that need specific conditions and nutrients to grow.

5. What is the theory upon which intermittent sterilization is based?

1. According to Farlex Partner Medical Dictionary, intermittent sterilization is “exposure to a temperature of 100°C (flowing steam) for a definite period, usually an hour, on each of several days; at each heating, the developed bacteria are destroyed; spores, which are unaffected, germinate during the intervening periods and are subsequently destroyed.”. The concept of destroying the highly resistant spores through intermittent sterilization is based on time. The theory believes that spores not destroyed in the first processing will be less resistant and destroyed after more sterilization cycles.

2. The sample is placed in the steam sterilizer or boiled to introduce moist heat for 10-20 minutes, which kills the vegetative cells and mist spores will likely remain. The concept of boiling is based upon the idea that spores are resistant to most sterilization procedures, hence the only way to get rid of them is to give them the opportunity to grow out and kill the cells before they reproduce their endospores. The sample is left for 24 hours for the spores to regenerate the vegetative cells, while it cools as the spores are resistant to high pressure and temperature. The next day, another sterilization cycle is done which destroys the vegetative cells that the endospores germinated after the first treatment. This process is done until all the bacteria and its endospores are no longer in the sample.

6. Is dry or moist heat more efficient as a sterilizing agent? Why?

1. Dry heat sterilization involves exposing a sample for sterilization in an oven at approximately 170°C for 2 to 3 hours under atmospheric pressure. This will cause the cell’s elements to oxidize and proteins to denature, killing the cell. This method of heat sterilization is used to sterilize hydrophobic compounds such as fats and oils, and substances susceptible to water damage such as metal instruments and powders. Steam sterilization is done in an autoclave, where the equipment can produce pressurized steam to create a high heat at 121°C. Sterilization takes only 15-30 minutes and during this time the cell membranes of the microbes hydrolyse and the proteins coagulate and denature, killing the microbial cells. It is best used for sterilizing culture media, liquids and heat-sensitive items.

2. Though both methods have their advantages, in terms of efficiency, moist heat sterilization is the better of the two as the steam produced in the autoclave is a good heat conductor, making it easier to penetrate and kill the microbes. Though dry heat sterilization has a lower initial cost, less time is used during sterilizing a sample with moist heat which results in long-term cost savings. It is also the most efficient in sterilizing culture media because with dry heat sterilization the liquid of the medium will boil off and damage the media.