INOCULATION OF CULTURE MEDIA

Questions:
1. What is the purpose of flaming in aseptic technique?

The purpose of flaming in aseptic technique is first, for the inoculating loop
or needle to be sterilized and this must be performed before and after the microbial
transfer. This heating eliminates any living organisms on the needle’s or loop’s
surface, thus preventing contamination. Also, heating particularly in test tubes with
bacteria, warms the bottle’s opening and induce air convection currents up and
away from it. The dust particles and other pollutants then kept out of the bottle by
the heated, rising air.

2. What are some signs of growth in a liquid medium?

Some of the signs that there is a growth in a liquid medium includes: the
change in the sample’s turbidity or the cloudiness or haziness of the fluid; the
subculture tends to become a semi solid medium; natural gas production (bubbles
in an inverted Durham tube or analysis of head space gas above the liquid for
changes); generation of acid (color changes in a pH sensitive indicator) and;
colonies suspended in liquid in a distinctive manner.

3. How can one maintain culture purity and prevent contamination?

Knowledge should be the first consideration in terms of handling cultures. It will

help you to manage well the bacteria in a safe and appropriate way. To be specific,
here are some ways how to maintain the culture’s purity and eventually prevent
contamination. First, is transferring to new media on a regular basis. Strains can
be kept alive by making a fresh culture from the previous stock culture on a regular
basis. Second, refrigeration. Pure cultures can be kept at a temperature of 0-4°C
in refrigerators or cold rooms. Because the metabolic processes of the
microorganisms are considerably slowed but not stopped, this approach is used
for a limited period of time (2-3 weeks for bacteria and 3-4 months for fungi). Third,
Paraffin Method/ preservation by overlaying cultures with mineral oil. This
procedure involves pouring sterile liquid paraffin over the culture's slant (slope)
and storing it upright at room temperature. The paraffin layer maintains anaerobic
conditions and prevents the medium from becoming dehydrated. Fourth,
Cryopreservation. Cryopreservation (freezing in liquid nitrogen at -196°C or in the
gas phase above liquid nitrogen at -150°C) aids in the long-term storage of pure
cultures. Lastly, Lyophilization (Freeze-Drying). Sublimation removes water and
other solvents from a frozen product during the freeze-drying process. Sublimation
is the process by which a frozen liquid transitions from a solid to a gaseous state
without passing through the liquid phase.
REFERENCES

Greenwood, M. (2019) Aseptic Techniques in Microbiology. News Medical Life Sciences.

https://www.news-medical.net/life-sciences/Aseptic-Techniques-in-
Microbiology.aspx

UK Standards for Microbiology Investigations (2020) Inoculation of culture media for

bacteriology. Public Health England.
https://assets.publishing.service.gov.uk/government/uploads/system/uploads/atta
chment_data/file/583859/Q_5i2.pdf

Fitzgerald, H., Wilber, P.G., Bentz, K., Peterson, A. (2018) Unit 3. Microbial Growth,
Aseptic Inoculation & Streak Isolation.

Britannica (2022) Inoculation. https://www.britannica.com/science/inoculation