CULTURE METHODS
CULTURE 2) Chocolate Agar
➢ A method of multiplying microbioal organisms by
allowing them to reproduce in a specific culture
medium under controlled laboratory conditions
➢ Basically culture, you are trying to grow microbial
organisms, you are trying to grow your own
organisms
CULTURE MEDIA
➢ Enriched media contain the nutrients required to
support the growth of a wide variety of organisms,
including some of the more fastidious ones
o Fastidious – they have complex set of
nutritional requirements needed to grow
(artehon nga kagaw); that’s why some
organisms can only grow in specific types of
media
➢ Are commonly used to harvest as many different
types of microbes as are present in the specimen
➢ These are optimized to grow as much as you need
with as little space (we grow these in petri dishes)
COMMON EXAMPLES OF CULTURE MEDIA
1) Blood Agar
o Blood agar is an enriched medium in which
nutritionally-rich whole blood supplements the
basic nutrients
o Usually, the blood used is 4% sheep’s blood
o Often used to grow fastidious organism and to
differentiate bacteria based on their hemolytic
properties
o Remember, this is blood, there is blood in this
agar, so if a particular microbial organim/
bacterium is hemolytic, you will see that there will
be results of hemolysis here; they will break down
the blood; you will see blank spots in your media
o Enriched with heat-treated blood (40-45°C), which
turns brown and gives the medium its distinct
brown color
o It’s not really chocolate; it’s still blood, sheep’s
blood usually; called chocolate agar because it’s
brown and looks like chocolate but it’s actually
blood
o Used for growing fastidious respiratory bacteria,
such as
➢ Haemophilus influenzae – causes pneumonia
➢ Neisseria meningitidis – causes bacterial
meningitis
CULTURE METHODS
➢ The technique used and the type of medium
selected depend on the nature of the investigation
Three (3) Purposes why we culture our own bacteria:
1) To raise a crop of cells of a particular species that is
on hand
- Let’s say, you want to test something on Staph.
areus and you only have a limit number of
Staph. aureus on hand; so you would like to
multiply their number so you have raised a
crop of Staph. aureus
2) To determine the numbers and types of organisms
present in a given material
- Example, you want to test your fellow
classmates about hygiene habits so you
swabbed their hands; you are trying to test the
different types and mount of bacteria present
in their hands at that given time; you are
testing for presence of bacteria
 CULTURE METHODS
3) To isolate a particular type of microorganism from
a natural source
- Let’s say, you have a contaminated water
sample (drinking water) with Legionella; so you
are trying to isolate the Legionella to extract it
for further testing; so you are trying to get it
from a particular sample for isolation
- Anaerobic, 3rd Plate – Acidic, 4th Plate – Basic,
GROWING CELLS OF A GIVEN SPECIES
1st purpose; You are trying to grow more of a given
bacteria/ organism
➢ Microorganism observed microscopically to be
growing in a natural environment may prove
exceedingly difficult to grow in pure culture in an
artificial medium
➢ Some parasites have never been cultivated outside
their hosts (because they need a living host to
thrive)
➢ A suitable medium can be devised by carefully
reproducing the conditions found in the organism’s
natural environment
Factors/ Conditions:
a) ph – some thrive in acidic, neutral, or basic/
alkaline environments
b) Temperature – some like it hot or cold
c) Aeration – some are aerobes, some are
anaerobes; some die when exposed to oxygen like
Clostridium tetani (causes tetanus)
d) Nutrients – like the Haemophilus influenza and
Neisseria meningitides, they like the blood agar
MICROBIOLIC EXAMINATION OF NATURAL MATERIALS
nd
2 purpose; we are trying to test a particular material
or particular specimen for the different microorganisms
present
➢ A given natural material (food or swab specimen)
may contain many different microenvironments
(remember, some are fastidious), each providing a
niche for a different species
➢ Plating a sample of the material under one set of
conditions will allow a selected group of forms to
produce colonies but will cause many other types to
be overlooked → plate out samples of the material
using as many different media and conditions of
incubation as is practicable
- Example: 1st Plate – Aerobic, 2nd Plate –
5th Plate – Neutral
- These 5 plates will produce different types of
microorganisms; so you will need as many
plates, as many sets of conditions as you can to
determine the different types of organisms
present in you origin material
➢ Solid media are used, and crowding of colonies is
avoided
- We avoid crowding the colonies because we
are trying to get as many conditions as
possible
- If you crowd the colonies, this will cause
competition between the colonies or
bacteria which will result to them dying out
➢ When you do this 2nd purpose, this is how you
should do it/ this is the normal site:
- A number of plates with varying conditions in
each
ISOLATION OF A PARTICULAR TYPE OF MICROORGANISM
3rd Purpose
➢ Done by selecting for the desired type
➢ Inoculation of the sample into medium that has
been made up for the purpose of favouring one
type of organism – enrichment culture
 CULTURE METHODS
- You are enriching it with the conditions needed
for that particular organism to survive
➢ Searching for a particular type of organism that is
part of a mixed population – selective or differential
media are used (selective or differential media is a
culture medium that inhibit the growth of
organisms other than the one being sought
- Example: you want to isolate Azotobacter;
Azotobacter is a common bacteria found in the
soil; Azotobacter likes nitrogen so your selective
or differential media will have to be rich in
nitrogen so that your Azotobacter will thrive
while the other organisms will not
- That is why it is called selective or differential
media because you are selecting a particular
bacteria and discarding the rest of the bacteria
Common Examples of Selective or Differential Media
Thayer-Martin Agar
o Used to isolate Neisseria gonorrhoeae, the
cause of gonorrhoea (a very common STI), from
clinical specimens
- This is a rectal swab form a patient infected
with Neisseria gonorrhoeae
- If you swab the specimen in a regular medium/
chocolate agar (left), there will be different
types of bacteria growing
- But if you swab it only on Thayer-Martin (right),
remember Thayer-Martin is a selective media
and only selects Neisseria and the rest will die
- Hence, only Neisseria is growing
PURE CULTURE
➢ A population of cells or muticellular organisms
growing in the absence of other species or types
➢ That is why it is called pure culture because you
only have the population of one particular cell or
organism
➢ Ideal gelling agent for most microbiologic media is
agar
➢ The media is called pure culture if you are only
growing one particular organism/ species
DIFFERENT TECHNIQUES OF PLATING
1) Streak Plate Technique
➢ Produces an isolated colony of an organism on the
agar plate
➢ Isolation of the organism is a must in a mixed
culture, especially if thorough study for the colony
morphology of a particular organism is needed
➢ Because some of the bacteria that we study have
characteristic colonies
- Ex. your Staphylococcus aureus is called as such
because aureus comes from the Greek word
aerus which means gold (also why the elemental
symbol for gold us Au); so your Staphylococcus
aureus will appear as golden colonies
How to do the Streak Plate Technique:
✓ You have your plate containing your agar
 CULTURE METHODS
✓ You have a wire loop, this is a loop of wire
connected to a handle
✓ What you do first is to light up your Bunsen
burner and sterilize your wire loop
✓ Once the wire loop is red-hot and glowing, you
let it cool off for a few seconds and then you dip
it into your specimen/ to your sample
✓ Once it is coated with your sample, you do a
streaking of your plate until it reaches about ½
of your plate
2) Pour Plate Method
✓ After which, you sterilize again your wire loop
over your Bunsen burner until it glows red-hot
and let it cool down again, and repeat the
process
How to do the Pour Plate Method:
✓ Dip it in your sample and streak again and again
and rinse and repeat for your last section
*some techniques of the streak plate will require 3
or 4 quadrants (but for this visual aid, it is only 3 )
➢ Basically, the purpose of this is; if after you
culture your organisms, it usually takes a day,
when you come back the next day, you examine
that all of the colonies that grow are identical
(meaning, that you have successfully isolated a
pure culture of a specific or particular
microorganism)
➢ If ever you examine your streak plate and you
see that there is one streak that looks different
from the other, that means you have
unsuccessfully isolated a pure culture (it means
your culture has been contaminated by another
species of microorganisms and you will have to
repeat your streak plate again until all colonies
on the streaks appear similar)
➢ Pour it on a plate
➢ Usually the method of choice for counting the
number of colony-forming bacteria present in a
liquid specimen
➢ We try to isolate one cell at a time and see if it
has the potential to form colonies
✓ We put our sample into a test tube with dilution
agent (usually water)
✓ We keep pouring it to different test tubes with
different levels of dilution
 CULTURE METHODS
✓ We increase the dilution until we isolate single
cells of bacteria (right) compared to multiple
cells (left)

*We try to isolate the most diluted form of the

organism because remember, we are trying to see if
they are capable of forming colonies

*we are trying to see if this single cell of bacteria

will form into a colony after culture (purpose of this
method)

3) Spread Plate Technique

➢ A technique to plate a liquid sample containing

bacteria so that the bacteria are easy to count
and isolate

How to do the Spread Plate Method:

✓ We have our bacterial dilution and we pour it

on our plate
✓ This is called spread plate because we are using
a spreader to spread it our evenly
✓ We flatten the sample; when we flatten the
sample, it is easier to count because from 3-
dimensional, we made it into 2-dimensional and
our microscopes are 2-dimensional (if you
spread it out and flatten it as much as possible,
then you made it 2D which makes it easier to
count the number of cells in that sample)
✓ Spread it out then you plate the incubated
sample until bacterial colonies grow on the
surface of the medium
✓ Then you count it through the microscope
 Staining methods
GRAM STAIN safranin which makes them appear pinkish under
the microscope
➢ This is to check whether a particular organism is
gram positive or gram negative ACID FAST STAIN
➢ This is for the easy identification of the specimen
and ruling out all plausible alternatives ➢ A.k.a Ziehl-Neelsen staining
✓ Gram positive: purple (crystal violet) ➢ Used to identify acid-fast bacteria
✓ Gram negative: pink (safranin) ➢ Acid-fast bacteria are those that retain
carbolfuchsin even when decolorized with
Four (4) Steps of Gram Staining: hydrochloric acid in alcohol
➢ Primary stain: Carbolfuchsin

Similar to Gram Staing EXCEPT:

✓ Primary stain is carbolfuchsin

✓ Uses (light) heat to affix the stain
✓ Decolorizing agent is acid alcohol (alcohol mixed
with acid)
✓ Counter stain is methylene blue
1) Application of Crystal Violet (purple dye)
Four (4) Steps of Ziehl-Neelsen Staining:
- Apply the crystal violet which is the purple dye
- the crystal violet is easily absorbed by your gram
positive organisms
- if your organism is positive, it will absorb this dye
to its cell wall
2) Application of Iodine (Mordant)
- Next is the application of mordant, your iodine
- This is to affix your stain
- If ever your crystal violet is absorbed by your
bacteria, this mordant affixes it to the cell wall of
the bacteria 1)Apply primary stain of carbolfuchsin for 30 seconds
3) Alcohol Wash 2)Heat fix cells to slide using flame
- Next is your decolorizer which is alcohol or 3)Decolorize with acid alcohol for 15-20 seconds
acetone 4)Apply counterstain of methylene blue for 30
- When the decolorizer is poured into your second then rinse excess stain
sample, it washes away the crystal violet that - If the bacteria is acid-fast, then it will not
WAS NOT ABSORBED by your bacteria absorb the carbolfuchsin and instead, absorb
- So it will appear colorless if IT IS NOT GRAM the methylene blue
POSITIVE ✓ Acid-fast Bacteria: pink (carbolfuchsin)
4) Application of Safranin (Counterstain) ✓ Non-Acid-Fast Bacteria: blue (methylene blue)
- Lastly is the application of your safranin which is
the counterstain
- Safranin is pinkish in color
- If the organism is gram negative, they will not
absorb the crystal violet (which was washed
away by the decolorizer) but instead absorb the
 Staining methods
NEGATIVE STAIN

➢ Staining the background with an acid dye, leaving

the cells contrastingly colorless
➢ Nigrosin is commonly used (black)
➢ Used for cells or structures that are difficult to stain
directly
➢ Hence, instead of staining the organisms, we stain
the background

Steps of Negative Staining:

- We see lophotrichous flagella here which means
there is flagella on the polar opposites; only at one
point
Apply the nigrosin to the glass
slide
CAPSULE STAIN

➢ Stains the capsule

Spread it out with another glass
slide
Spread it out until it is almost
completely stained
➢ Capsules are usually demonstrated by the negative
staining procedure or modification of it
➢ Welch Method – involves treatment with hot crystal
violet solution followed by a rinsing with copper
sulphate solution; a modification of the negative
staining

➢ When you look at it under the miscroscope,

everything is black except for your organism

FLAGELLA STAIN
Flagella is the structure in bacteria for locomotion/
movement
➢ Used to identify whether an organism is flagellated
➢ Flagella are too fine/ too small to be visible in the
light microscope
➢ Cells are treated with an unstable colloidal
suspension of tannic acid salts , causing heavy
precipitate to form on the cell walls and flagella,
making it more easy to identify
- After your organisms are stained/ capsule stained,
there will appear like this; the capsule is easily
identifiable around your bacteria
SPORE STAIN
➢ The spore wall is relatively impermeable, difficult to
penetrate
➢ but dyes (malachite green or carbolfuchsin) can be
made to penetrate it by heating the preparation
which prevents decolorization of the spore by a
period of alcohol treatment sufficient to decolorize
 Staining methods
vegetative cells, after which they are
counterstained
➢ When you heat the malachite green or
carbolfuschin, it makes it penetrate the spore wall
and decolorization is prevented during the
decolorization phase of the staining
➢ After which they are counterstained as a contrast
➢ You will know it is the spore because it absorbed
the primary stain

- Looks like this under the microscope

- Spores are the ones that appear

NUCLEOID STAIN
Actually in our books but not performed in the lab
because it can only be appreciated with an electron
microscope

➢ You are basically staining the nucleoid

➢ Can be appreciated under an electron microscope
➢ It will look like this; you are staining the nucleoid
inside the body of the bacteria