This document provides instructions for preparing bacterial culture media and growing bacteria in the laboratory. It discusses three key steps: 1) preparing suitable culture media with nutrients and minerals to support bacterial growth, 2) sterilizing media and containers to remove other organisms, and 3) adjusting the pH of the media. Specific recipes are provided for preparing nutrient agar and nutrient broth to culture bacteria. The document also describes inoculating and incubating the media, staining bacterial samples, and examining them under a microscope.
2. Introduction
• Culture media are mediums that provide essential nutrients and
minerals to support the growth of microorganisms in the laboratory
• In the process of culturing bacteria, there are three steps
1. The preparation of a suitable culture medium
2. The initial removal of other organisms from the medium
and its containers by sterilization
3. Adjustment of pH of the medium
3. PREPARATION OF MEDIA
• The basis for almost all of the bacteriological media is some kind of
extract of meat (broth), which provides most of the substances
required for bacterial growth
• The media may be solid or in liquid form
• Gelatin is an albumin-like material derived from tendons and cartilage
• Agar is prepared from dried seaweed
5. Preparation
1.Suspend 28g of nutrient agar powder (CM0003B) in 1L of distilled
water.
2.Mix and dissolve them completely.
3.Sterilize by autoclaving at 121°C for 15 minutes.
4.Pour the liquid into the petri dish and wait for the medium to solidify
5.When these ingredients are dissolved in a steamer
6.The pH is adjusted to between 7.2-7.6
6. Nutrient Broth
• The formula for the nutrient broth is the same, except that agar is not
added in it
• Therefore, the medium remains in liquid form
• It is dispensed in sterile, screw-capped tubes
7. Adjustment of pH
• The pH of a medium is a matter of great importance if good growth of
the organisms is to be obtained and it must be adjusted before the
medium is used
• The amount of N/10 HCl or N/10 NaOH that is to correct the pH of
the 5 ml sample is then determined by titration
8. The inoculation of culture media
1. Sterilize the wire loops and other instruments in a flame, before
and after use. Protect yourself from the dangers of aerosols. Masks
should be used.
2. Flame the mouth of the culture bottles and tubes after removing
and before replacing their covers.
3. De-contaminate the table before you start working and after you
have finished the day‘s work.
9. Continue…
4. Air currents should be reduced to a minimum by closing windows
and doors and restricting the movement of people in the room.
5. During the inoculation, a culture medium should be uncovered for
only a few seconds.
6. Place the lighted Bunsen burner and inoculating instruments to the
right of the bench, and cultures and media to the back and the left. (If
the operator is right-handed).
10. CONTINUE…
7. Media for seeding should be labelled, indicating the inoculum and
the date with glass-marking pen, before seeding the plate.
8. Labelling should be on the bottom of the petri dishes, on tubes and
on bottles rather than on lids or caps.
9. During inoculation, the right hand holding the inoculating
instrument charged with the culture material from the specimen
should be moved as little as possible and the left hand should bring the
media to it.
12. Incubation
• Turn the plates upside down and put them in a warm place.
• The ideal temperature for incubation is 32° C or 90° F.
• Bacterial growth should start to become visible in about 2 -3
days.
13. Preparation of slide
• . Place one needle of solid bacterial growth or
two loops of liquid bacterial growth in the center
of a clean slide.
• If working from a solid medium, add one drop
(and only one drop)of water to your specimen
with a water bottle. If using a broth medium, do
not add the water.
14. Continue…
• Now, with your inoculating loop, mix the
specimen with the watercompletely and
spread the mixture out to cover about half
of the total slide area.
• Place the slide on a slide warmer and wait
for it to dry. The smear is now ready for
the staining procedure
15. Staining
• The Gram staining process includes four basic steps, including:
1.Applying a primary stain (crystal violet).
2.Adding a mordant (Gram’s iodine).
3.Rapid decolorization with ethanol, acetone or a mixture of both.
4.Counterstaining with safranin.
17. Microscopic Examination
• If the bacteria was colored purple, it means you likely have a
Gram-positive infection.
• If the bacteria was colored pink or red, it means you likely have
a Gram-negative infection
