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General Technique for Bacteria and Fungi Cultures
Caution: Do not work with pathogenic (disease-causing) organisms unless you have had sufficient
training and experience in their handling. Treat all bacteria as being pathogenic, both to establish
proper work habits and because of the chance (however small) of a culture are inadvertently becom-
ing contaminated by a pathogen. Even non-pathogens are pure cultures and are heavily concentrated,
and, as such, should be handled with care.
1. Sterilization
a. Media and glassware: Use an autoclave or pressure cooker at 15 lbs. for 15 min. at 121°C.
b. Glassware: Dry heat in oven at 160°C for at least two hours.
2. Wear a clean lab coat, smock, or apron to protect clothing and to reduce possible contamination
of cultures.
3. While in the lab, avoid any hand to mouth operations, such as eating, smoking, or licking
adhesive labels.
4. Wash hands thoroughly with soap and water, both before and after working with cultures.
5. Keep work surface clear of any unnecessary objects (e.g., books, purses, etc.)
6. Wash work surface with a capable disinfectant, such as 10% Lysol, 70% alcohol, or household
bleach both before and after working with cultures.
7. Culture transfers
The only articles of equipment needed to make a transfer from the initial culture to a tube of sterile
medium are a Bunsen burner or similar heat source, and an inoculating loop, needle, or sterile swab.
a. Hold both tubes in the left hand (Figure 6).
Figure 6
b. With the needle in the right hand, pass the entire
length of the wire through the flame until it has all
turned red hot (Figure 7).
Figure 7
c. While still holding the needle, QUICKLY remove the caps or plugs
from the tubes, holding them between the fingers of the right
hand (Figure 8). Flame the mouths by passing them two or three
times through the burner flames (Figure 9). Hold these tubes
almost parallel to the table top if they contain a broth. This will
reduce the possibility of air-borne contaminants.
Figure 8
