Microbiology

Culture:
1. Streak Plate

2. Pour Plate
3. Spread Plate
Comparison:

Streak Plate Pour Plate Spread Plate

Volume of No definite/exact 1.0 mL or 0.1 mL 0.1
inoculum amount(Only a
Loopful)

Purpose For isolation of Count the number of Count the number of

colonies only colonies(number colonies(number
of microorganisms) of microorganisms)

Equipment Petri dish Petri dish Petri dish

(Glasswares)
Alcohol Lamp Alcohol Lamp Alcohol Lamp
Wire Loop Pipette Pipette
Test tubes Test tubes
 Glass Rod / L-rod
Type of Colonies Surface Colonies Either Surface/ Surface Colonies
SubsurfaceColonies

Advantage * For isolation of *For Quantification of * Cultures are never

bacterial cultures colonies in solid medium exposed to 45oC
melted agar
* Has distinct *Allows the growth and
temperatures.
separate colonies quantification of
microaerophiles(Sufficient
oxygen supply)
Disadvantage * Higher * Picking subsurface * More microbes,
Probability of colonies would interrupt presence of more
Contamination other colonies by digging colony forming
prior to isolation out of the agar units.
* Microbes must withstand
the agar temperature

4. Drop Plate (Serial Dilution)

- Dilute the sample inoculum into different dilutions
- Expel diluted solutions in three to five evenly spaced 10 µl drops onto the quadrant of
one of the petri plates that have been labeled for that particular dilution of the sample.
- Need to wait until the solutions fully absorbed into the agar before store inside
incubator to incubate.
- drop plate is commonly use in lab for counting the CFU/mL (Colony-Forming Unit)
5. Agar Slant
6. Agar Deep
Agar Slant Agar Deep

Larger surface area Smaller surface area

By streaking organism on the surface of the By stabbing organism into the agar
slant
Used for maintenance and preservation of Used for storage and for studying the
pure cultures for subculture purposes gaseous environment for Mos

7. Nutrient Broth

Nutrient broth is typically made of a powdered beef extract that contains peptones
(broken down proteins). The powder is dissolved in water, put in test tubes, and sterilized.
Broth is convenient, as most bacteria will grown in this type of medium, even those with
widely different aerotolerances (oxygen requirements).
Disadvantage: bacterial colonies do not form in a liquid suspension.
Culture Tools:
1. Wire Loop/ Inoculating needle
2. Micro pipettes
3. Sample inoculum
4. Solutions for dilution
5. Glass spreader
6. Bunsen burner

Culture Media:
 Suitable for Student
Type of Agar Brief Description
Use?

No, due to potential for

Contains blood cells from an animal (e.g. a sheep); most bacteria will grow on this
Blood Agar contamination from
medium.
human contact.

Comprised of sheep blood that provides the X and V factors necessary

for Haemophilus growth, this is a nutrient medium which is used in culturing No, due to potential for
Chocolate Agar fastidious organisms such as Haemophilus species and Neisseria. Chocolate agar, contamination from
however, does not reveal hemolysis data, so species differentiation among the human contact.
members of Haemophilus must be performed in another manner.

This is an agar upon which only Gram-negative bacteria can grow. What is more is
that E.coli will grow into red colonies, as there is a pH indicator present. It should
be mentioned that MacConkey agar powder comes in two versions: one with the
MacConkey
sugar lactose in it, and one without any added sugars. Since E.coli ferments sugars No, due to selectivity.
Agar
to acids (thus the red color), one can add one of the many different kinds of sugars
to this sugar-free MacConkey agar and see if red colonies develop. If you get red
colonies, you know the E.coli strain you are using can use that sugar.

Non-nutrient Usually not suitable for growing bacteria. However, may be used for growing other
No.
Agar microorganisms.

Will grow the largest number of different types of microbes - fungi and bacteria.
Nutrient Agar Yet, not all bacteria can grow on these. Some find it too rich, and others find it Yes.
deficient. The nutrient in this is beef broth, and some extracts from yeast.

Xylose lysine deoxycholate agar. It is used for the culture of stool samples, and
contains two indicators. It is formulated to inhibit Gram-positive bacteria, while the
XLD Agar No, due to selectivity.
growth of Gram-negative bacilli is encouraged. The colonies of lactose fermenters
appear yellow.

Brilliance MRSA 2 Agar can be inoculated from a screening swab taken from
hospital patient or staff, from an isolated colony or from a liquid suspension. MRSA No, Brilliance MRSA 2
Brilliance MRSA
grows as blue colonies which are very easy to read against the light coloured, Agar is for in vitro
2 AGAR
opaque background. Accuracy minimizes costs, by helping to ensure that only those diagnostic use only, by
in need receive what can be costly treatment. trained individuals
Positive control : Staphylococcus aureus

TCBS Agar is used for the isolation of Vibrio cholerae and other
Thiosulfate- enteropathologic Vibrio (in particular Vibrio parahaemolyticus) in fish, seafood and
Citrate-Bile biological samples of animal origin.
Salts-Sucrose TCBS agar has also been used to control outbreaks of the crown-of-thorns seastar
(TCBS) Agar (Acanthasterplanci).
(https://microbiologyinfo.com/thiosulfate-citrate-bile-salts-sucrose-tcbs-agar-
composition-principle-uses-preparation-and-colony-morphology/)

https://www.sciencebuddies.org/science-fair-projects/references/grow-microbes-agar
COMPARISON BETWEEN GRAM POSITIVE AND GRAM NEGATIVE BACTERIA
Gram-negative Bacteria Gram-positive Bacteria
Gram reaction Can be decolourized to accept counter stain Retain crystal violet dye and stain dark violet
(Safranin or Fuchsine); stain red or pink, they or purple, they remain coloured blue or purple
don't retain the Gram stain when washed with with gram stain when washed with absolute
absolute alcohol and acetone. alcohol and water.
Peptidoglycan layer Thin (single-layered) Thick (multilayered)
Teichoic acids Absent Present in many
Periplasmic space present Absent
Outer membrane Present Absent
Lipopolysaccharide High Virtually none
(LPS) content
Lipid and High (due to presence of outer membrane) Low (acid-fast bacteria have lipids linked to
lipoprotein content peptidoglycan)
Toxins produced Primarily Endotoxins Primarily Exotoxins
Resistance to Low High
physical disruption
Inhibition by basic Low High
dyes
Susceptibility to Low High
anionic detergents
Resistance to drying Low High
Cell wall The cell wall is 10 nanometer thick; two The cell wall is 15-80 nanometer thick; single
composition layered. Lipid content is 20-30% (high), Murein layered. Lipid content of the cell wall is low ,
content is 10-20% (low). whereas Murein content is 70-80% (higher).
Mesosome Mesosome is less prominent. Mesosome is more prominent.
Antibiotic More resistant to antibiotics. More susceptible to antibiotics
Resistance

EXTRA: Precautions:
1. Everything should be work around the bursen inside the sterilize zone to reduce
the contamination.
2. When working with pour plate method, make sure that the molten agar
temperature is not too high so it won’t kill the bacteria.
3. Flame the wire loop or inoculating needle until red hot every time before and after
using.
4. Make sure to cool down the wire loop before withdraw the culture.
5. Flame the neck of bottles of test tubes after opening and before closing the cap.
6. Mind with the first and second stop of pipettes.
7. Seal the petri dish before incubate.
 Zone of Inhibition Test = Kirby-Bauer Test

= a qualitative method used clinically to measure antibiotic resistance and industrially to

test the ability of solids and textiles to inhibit microbial growth.

If the bacterial or fungal strain is susceptible to the antimicrobial agent, then a zone of
inhibition appears on the agar plate, such as on the agar plate on the left-hand side of the
photo below. If it is resistant to the antimicrobial agent, then no zone is evident, such as
on the agar plate on the right-hand side of the photo below.

The size of the zone of inhibition is usually related to the level of antimicrobial activity
present in the sample or product - a larger zone of inhibition usually means that the
antimicrobial is more potent.
STRENGTHS OF ZONE OF INHIBITION TESTING

 fast and inexpensive

 well suited for determining the ability of water-soluble antimicrobials to inhibit
the growth of microorganisms.
 A variety of antimicrobial product types can be tested using this method. Liquids,
coated antimicrobial surfaces, and antimicrobial-impregnated solid products can
all be tested for their ability to produce a zone of inhibition.

WEAKNESSES OF ZONE OF INHIBITION TESTING

 do not necessarily indicate that microorganisms have been killed by an

antimicrobial product - just that they have been prevented from growing.
 Microbial growth agars may interfere with the function of some antimicrobial
agents.
 zones of microbial inhibition do not always have clear or regular boundaries.
 The method is not classically quantitative (though sometimes the diameter of the
zones of inhibition are measured and recorded).