Pre - Report

Microbe Smear and Colony Observation on Solid Medium

2016 / 03 / 28
Section 1 / 2015101109 / Rahman Tony Nur

1. Title
Microbe Smear and Colony Observation on Solid Medium

2. Purpose
a. Learning the method of preparing solid medium.
b. Learning universal microbial cultivation technique on solid medium.

3. Theory
a. LB (Luria Bertani) medium
LB is a widely used bacterial culture medium. The agar form of the medium should be
designated LA but it is often referred to as LB. Although originally developed for
bacteriophage studies and Shigella growth, LB subsequently became the medium of choice
for growth of Escherichia coli and other related enteric species. LB has also been used as a
general-purpose bacterial culture medium for a variety of facultative organisms. In the
undergraduate microbiology teaching labs, LB is sometimes used as the growth surface when
attempting to analyze bacterial colony morphology
a. Liquid medium
Liquid medium are water-based solutions that do not solidify at temperatures above
freezing & that tend to flow freely when the container is tilted. Termed broths, milks, or
infusions, they are made by dissolving various solutes in distilled water. Growth occurs
throughout the container & can then present a dispersed, cloudy, or flaky appearance. A
common laboratory medium, nutrient broth, contains beef extract & peptone dissolved in
water. Methylene blue milk & litmus milk are opaque liquids containing whole milk and
dyes. Fluid thioglycollate is a slightly viscous broth used for determining patterns of
growth in oxygen.
b. Solid medium (with the addition of agar)
Solid media provide a firm surface on which cells can form discrete colonies and are
advantageous for isolating and culturing bacteria and fungi. The most widely used and
effective of these agents is agar, a polysaccharide isolated from the red Gelidium. The
benefits of agar are numerous. It is solid at room temperature, and it melts at 100 . Once
liquefied, agar does not resolidify until it cool to 42 , so it can be inoculated and poured
in liquid form at 45 to 50 that will not harm the microbes or the handler (body
temperature is about 37). Agar is flexible, moldable, and provides a basic framework to
hold moisture and nutrients. Another useful property is that it is not readily digestible and
thus not a nutrient for most microorganism.
Any medium containing 1% to 5% agar usually has the word agar in its name. Nutrient
agar is a common one. Like nutrient broth, it contains beef extract and peptone, as well as
1.5% agar by weight. Although gelatin is not nearly as satisfactory as agar, it will create a
reasonably solid surface in concentrations of 10% to 15%. The main drawback for gelatin
is that it can be digested by microbes and will melt at room and warmer temperatures,
leaving a liquid.
c. Composition of LB medium
NaCl
10g/L (1%, w/v)
Tryptone
10g/L (1%, w/v)

b. Streaking & Spreading

Yeast Extract
Agar (solid medium)

5g/L (0.5%, w/v)

15g/L (1.5%, w/v)

 The streaking method

is a rapid qualitative
isolation method. It is
essentially a dilution
technique
that
involves spreading a
loopful of culture
over the surface of an
agar plate. Although
many
types
of
procedures
are
performed, the fourway, or quadrant,
streak is described. It
is important for us to
work with bacteria that are genetically identical. By streaking for single colonies, one
isolated colony will form from a single bacterial cell and thus the colony is genetically
identical. Furthermore, streaking helps to ensure that single colonies can be isolated 8 - 12
hours later.
The spreading technique requires that a previously diluted mixture of microorganisms be
used. During inoculation, the cells are spread over the surface of a solid agar medium with a
sterile, L-shaped bent rod while the Petri dish is spun on a lazy-Susan turntable.

4. Experimental Method (Reagent & Apparatus)

a. Reagent & Apparatus
1. Eschericia coli
2. Loop
3. Alcohol lamp
4. Petri dish
5. pH meter
6. Weighing scale, quantitative spoon
7. Autoclave

8. 1N HCl, 1N NaOH
9. Materials for medium manufacturing
trypton 10g/l
yeast extract 5g/l
NaCl 10g/l
Agar 15g/l
Deionized water 1.0l

b. Solid medium manufacturing

1. Add following into a 250 ml or 300 ml erlenmeyer flask.
a. NaCl 1g/100 ml
d. Deionized water 100ml
b. Tryptone 1g/100ml
e. Agar 1.5g/100ml
c. Yeast Extract 0.5g/100ml
2. Mix gently - Agar cannot be dissolved in the water at room temperature. Remaining agar
particles will be dissolved during the autoclaving.
3. Cover the flask mouth with aluminum foil and label using steam indicator tape.
4. Autoclave the mixture at 121 for 20 minutes.
5. After the pressure has been lowered, chill the medium to around 50 using cool water.
6. Sterilize the bench using 70% ethanol.
7. Pour appropriate amount of medium to each Petri dish. (~25mL).
*Note: Pour the medium slowly to the center of the Petri dish. Make it sure that the
medium has been spread on the entire plate.

*Note: If the bubble has been generated, remove them quickly using sterilized tips.
8. Place the lid on each plate and allow them to cold down to room temperature for 10-20
minutes (until the medium gets solidified) and then invert the plates. Incubate the LB agar
plates at 37 for overnight.
9. Label the bottom of the plates with date. Seal the plates with plastic bags or parafilm and
store at 4.

c. Streaking
1.
2.
3.
4.
5.

6.
7.
8.
9.

10.

Sterilize the bench with 70% ethanol.

Prepare LB agar plates at room temperature.
Label the bottom of the plates with the strain name and the date.
Keep your table sterilized by working near a flame.
Get a single colony from plate using a flame-sterilized loop.
*Note: If you use a wire loop, you can sterilize it by passing it through a flame. Just
be sure to allow enough time to cool the loop before getting a colony.
Gently streak the bacteria over a section of the plate, as shown in the diagram (2.
Streaking) to create "zone ".
Using the flame-sterilized loop, drag through "zone " and streak the bacteria over a
second section of the plate to create "zone ".
Repeat the procedure to create "zone " and "zone ".
Incubate the plate for overnight (12-18 hours) at 37
Observe single colonies.

c. Spreading
1. Sterilize the table with 70% ethanol.
2. Label the petri dishes.
3. Apply appropriate amount of pre-cultured broth on the LB agar plates using a
micropipette and micropipette tips.
4. Dip a glass spreader into ethanol and then pass it through flame to sterilize the
spreader. After the flame is extinguished, touch to the medium to make sure it was cool
down.
5. Spread rup the spreader smoothly on the agar plate. (Drag it back and forth, rotate...).
6. Flame-sterilize the spreader for the next use.
7. Incubate the plates "UPSIDE-DOWN" at 37 for overnight.
8. Observe the colony formation.

5. Reference
1. James G. Cappuccino, Natalie Sherman; Microbiology A Laboratory Manual 8 th Edition; Person
Benjamin Cummings; 2008; p13-14
2. Kathleen Park Talaro; Foundations in Microbiology 7th Edition; McGraw-Hill; 2009; p60-63
3. http://www.microbelibrary.org/component/resource/laboratory-test/3031-luria-broth-lb-andluria-agar-la-media-and-their-uses-protocol
4. http://delliss.people.cofc.edu/virtuallabbook/StreakPlates/GoodStreak.html