Preparation of culture media and cultivation of microorganism

1. Laboratory Activity No. 5
Preparation of Culture Media
and
Cultivation of
Microorganisms

2. One that contains more than one
type of organism growing in a
sterile medium, such as agar. The
mixed culture can include multiple
species of viruses, fungi bacteria
and parasites, which may or may
not live in harmony with one
another, sharing the available
resources.
Mixed
Culture

3. A culture in which bacteria from
a foreign source have infiltrated
the growth medium.
Contamination may arise from
the operator and the laboratory
environment, from other cells
used in the laboratory, and from
reagents. Some infections may
present a risk to laboratory
workers: containment and
aseptic technique are the key
defense against such risks.
Contaminated
Culture

4. Laboratory culture containing a single species
of organism. A pure culture is usually derived
from a mixed culture (one containing many
species) by transferring a small sample into
new, sterile growth medium in such a manner
as to disperse the individual cells across the
medium surface or by thinning the sample many
fold before inoculating the new medium. Both
methods separate the individual cells so that,
when they multiply, each will form a discrete
colony, which may then be used to inoculate
more medium, with the assurance that only one
type of organism will be present. Isolation of a
pure culture may be enhanced by providing a
mixed inoculum with a medium favoring the
growth of one organism to the exclusion of
others
Pure Culture

5. Streptococcus are usually isolated on
Blood agar. Blood agar is one of the
most commonly used media in a
clinical lab. It consists of an enriched
agar base (Tryptic Soy agar) to which
5% sheep red blood cells have been
added. Blood agar is commonly used
to isolate not only streptococcus, but
also staphylococcus and many other
pathogens. Besides providing
enrichments for the growth of fastidious
pathogens, Blood agar can be used to
detect hemolytic properties.
Streptococcus sp.
in Blood Agar
Plate

6. Chocolate agar (CHOC) or chocolate
blood agar (CBA) is a nonselective,
enriched growth medium used for
isolation of pathogenic bacteria. It is a
variant of the blood agar plate,
containing red blood cells that have
been lysed by slowly heating to 80 °C
giving the medium a chocolate brown
color reason why it is called chocolate
agar. CHOC agar is used for growing
fastidious respiratory bacteria like
Haemophilus influenzae and Neisseria
meningitidis and gonorrhoeae which
Neither of these species is able to grow
on Sheep Blood Agar.
Neisseria sp. in
Chocolate Agar
Plate

7. Selenite F Broth is the medium used for the
selective enrichment of Salmonella spp from
both clinical and food samples. It is a buffered
Lactose Peptone Broth to which Sodium
Biselenite is added as the selective agent.
Selenite-F Broth was devised by Leifson, who
demonstrated that selenite was inhibitory for
coliforms and certain other microbial species,
such as fecal streptococci, present in fecal
specimens, and thus, was beneficial in the
recovery of Salmonella species. He found that
the inhibited strains would eventually
breakthrough, but if subcultures were made
from the enrichment broth after 8–12 h
incubation, the isolation of Salmonella was
possible without overwhelming growth of
many members of the intestinal flora.
Salmonella sp. in Selenite
F Broth

8. Mannitol Salt Agar (MSA) is a selective and
differential medium. The high concentration
of salt (7.5%) selects for members of the
genus Staphylococcus, since they can
tolerate high saline levels. Organisms from
other genera may grow, but they typically
grow very weakly.
MSA also contains the sugar mannitol and
the pH indicator phenol red. If an organism
can ferment mannitol, an acidic byproduct
is formed that will cause the phenol red in
the agar to turn yellow. Most pathogenic
staphylococci, such as Staphylococcus
aureus, will ferment mannitol. Most non-
pathogenic staphylococci will not ferment
mannitol.
Staphylococcus Aureus in
Mannitol Salt Agar

9. Salmonella Shigella (SS) Agar is
moderately selective and differential
medium for the isolation, cultivation and
differentiation of Salmonella spp. and
some strains of Shigella spp. SS Agar is a
modification of the Desoxycholate Citrate
Agar. It is recommended for testing clinical
specimens and food testing for the
presence of Salmonella spp. and some
Shigella spp. Shigella do not ferment
lactose or produce hydrogen sulfide gas,
so the resulting colonies will be colorless.
Salmonella will not ferment lactose, but
produce hydrogen sulfide (H2S) gas. The
resulting bacterial colonies will appear
colorless with black centers.
Shigella in Salmonella-
Shigella Agar Plate

10. Eosin Methylene Blue (EMB) agar is both
selective and differential culture medium. It
is a selective culture medium for Gram-
negative bacteria (selects against Gram-
positive bacteria) and is commonly used
for the isolation and differentiation of
coliforms and fecal coliforms.
EMB media assists in visual distinction
Escherichia coli, other nonpathogenic
lactose-fermenting enteric gram-negative
rods, and the Salmonella and Shigella
genera.
E.coli in Eosin Methylene
Blue Agar Plate

11. MacConkey agar is an indicator, a
selective and differential culture medium
for bacteria designed to selectively isolate
Gram-negative and enteric (normally
found in the intestinal tract) bacilli and
differentiate them based on lactose
fermentation
Klebsiella pneumonia colonies appear as
large shiny and dark pink in color. Two
distinguishing characteristics are lactose
fermentation on the medium and the
viscosity of the colonies. Encapsulated
strains of Klebsiella spp. are also mucoid
in appearance, which is a characteristic of
the strains of this genus.
Klebsiella Pneumoniae in
Mac Conkey Agar Plate

12. Bordet-Gengou agar is a type of agar plate
optimized to isolate Bordetella, containing
blood, potato extract, and glycerol, with an
antibiotic such as cephalexin or penicillin
and sometimes nicotinamide. The potato
extract provided nitrogen and vitamins,
and potato starch absorbed fatty acids
present in nasal secretions or collection-
swab cotton that inhibited growth; glycerol
was a carbon source. Medical
Microbiology, 4th edition states that
Regan-Lowe medium(containing charcoal,
blood, and antibiotic) has replaced Bordet-
Gengou medium as the medium of choice
for routine Bordetella pertussis incubation.
Bordetella Pertusis in
Bordet Gengou Agar Plate

13. Cetyltrimethylammonium bromide,
a quaternary ammonium, cationic
detergent, is the component of
Cetrimide Agar which allows for the
selective isolation of Pseudomonas
aeruginosa.
Cetyltrimethylammonium bromide,
when in contact with bacteria,
causes the release of nitrogen and
phosphorous from the bacterial
cell. Organisms other than P.
aeruginosa are unable to withstand
this germicidal activity.
Pseudomonas Aeruginosa
in Cetrimide Agar Plate

14. The Löwenstein–Jensen medium,
more commonly known as LJ
medium, is a growth medium specially
used for culture of Mycobacterium
species, notably Mycobacterium
tuberculosis.
When grown on LJ medium, M.
tuberculosis appears as brown,
granular colonies (sometimes called
"buff, rough and tough"). The medium
must be incubated for a significant
length of time, usually four weeks,
due to the slow doubling time of M.
tuberculosis (15–20 hours) compared
with other bacteria.
Mycobacterium
Tuberculosis in Lowenstein-
Jensen Medium

15. Thayer-Martin agar medium is a selective
medium used for the isolation of
gonococci (Neisseria gonorrheae) from
specimen containing a mixed flora of
bacteria and/or fungi e.g. urogenital
specimen. Modified Thayer-Martin (MTM)
agar is a GC agar base containing
vancomycin, colistin, nystatin, and
trimethoprim lactate (VCNT). Selective
isolation of Neisseria gonorrhoeae is
achieved with suppression of most other
gram-negative diplococci, gram-negative
bacilli, gram-positive organisms, and
yeast.
Thayer-Martin medium contains
antimicrobials which inhibit the growth of
organisms other than N. gonorrhoeae.
Neisseria sp. in Thayer
Martin Agar Plate

16. Potato Dextrose Agar (PDA) is used
for the cultivation of fungi. Potato
Dextrose Agar (PDA) is a general
purpose medium for yeasts and molds
that can be supplemented with acid or
antibiotics to inhibit bacterial growth. It
is recommended for plate count
methods for foods, dairy products and
testing cosmetics. PDA can be used
for growing clinically significant yeast
and molds. The nutritionally rich base
(potato infusion) encourages mold
sporulation and pigment production in
some dermatophytes.
Molds /Yeast in S Aboraud’s
Potato Dextrose agar Plate

17. TCBS Agar is prepared according to the formula
developed by Kobayashi. It is highly selective for the
isolation of V. cholerae and V. parahaemolyticus , in
addition to other Vibrio spp. TCBS has a very high
pH (8.5-9.5) which suppresses growth of intestinal
flora other than Vibrio spp. The medium consists of
plant and animal proteins, a mixture of bile salts, one
percent sodium chloride, sodium thiosulfate, ferric
citrate, sucrose, and yeast extract. The bile salts
inhibit growth of gram-positive microorganisms; one
percent sodium chloride is incorporated into the
medium to provide optimum growth and metabolic
activity of halophilic Vibrio spp.; sodium thiosulfate
provides a source of sulfur and also acts in
combination with ferric citrate to detect the
production of hydrogen sulfide; sucrose serves as
the fermentable carbohydrate that, with the help of
bromothymol blue and thymol blue indicators, allows
for the differentiation of those Vibrio spp. which
utilize sucrose.
Vibrio Cholerae in Thiosulfate
Citrate Bile salts (TCBS) Agar
Plate

18. Citrate agar tests the ability of organisms to
utilize citrate as a carbon source. Citrate
agar contains sodium citrate as the sole
source of carbon, ammonium dihydrogen
phosphate as the sole source of nitrogen,
other nutrients, and the pH indicator
bromthymol blue. This test is part of the
IMViC tests and is helpful in differentiating
the Enterobacteriaceae .
When Citrate agar is inoculated with
Escherichia coli , the medium remains green.
This is a negative result for the citrate test
When Citrate agar is inoculated with
Salmonella typhimurium , the medium turns
royal blue. This is a positive result for the
citrate test.
Escherichia Coli in
Citrate Agar Plate

19. The selective agents in the agar include
lithium chloride (LiCl), taking advantage of
listeria’s high salt level tolerance, as well
as anti-microbial compounds (ceftazidime,
polymyxin, nalidixic acid & amphotericin).
Enterococci and some Gram-negative
bacteria cannot tolerate high salt levels.
The LiCl content in Solus Agar-OA
therefore inhibits the growth of these
organisms. However, Staphylococcus and
Bacillus spp. are tolerant to LiCl, hence the
need for other selective agents.
Listeria Monocytogenes
in Listeria Agar Plate

20. 1. When the plates are incubated in the normal position, water evaporation from
media occurs. These vapors condense on the lid of the Petri dish and drops fall on
the colonies developed on the media surface. This causes colony mix-up with each
other and spread throughout the plate surface. This creates the problem in
counting and proper determination of microbial count.
2. The evaporation of water from media can cause media dryness that can affect the
microbial growth but when the plates are incubated in an inverted position, the rate of
evaporation decreases that results in proper microbial growth. Therefore, Petri dishes
with media can also be stored for a longer period in an inverted position.
3. The lid of Petri dishes may contain any contamination that spreads on the media
and grows with the sample microbes. This may create an error in microbial count
determination.
4. It is easy to handle the inverted Petri dishes because the lid of the Petri dish may
open during handling when incubated in normal position and it may cause
contamination from air. We label the Petri dishes at the bottom part because lid may
exchange with other Petri dishes creating confusion and inverted position makes it
easy to read the labeling of Petriplates.
Why are petri dishes incubated and when
cool/solidify are turn in an inverted position?

21. When sterilizing glassware such as bottles, petri dishes and test tubes,
dry heat is required and this is carried out in a hot air oven. The ideal temperature
of the oven needs to reach is at least 160°C and the contents need to be
regulated at this heat for 45 to 60 minutes. The contents must not be removed
from the oven immediately as a slow cooling period is necessary – ideally when
the temperature has reduced down to 50°c, but no less. The reason for the
gradual cooling period is to avoid the cracking of glassware as well as preventing
air (that could potentially contain contaminating organisms) entering the oven.
When microbiological media has been made, it still has to be sterilized
because of microbial contamination from air, glassware, hands, etc. Within a few
hours there will be thousands of bacteria reproducing in the media so it has to be
sterilized quickly before the microbes start using the nutrients up. Autoclave is
used for wet heat sterilization. Although there are other forms of sterilization,
autoclave is mostly the quickest. The sterilization process in autoclave can
guarantees that the medium will stay sterile unless exposed to contaminants by
less than adequate aseptic technique to exposure to air.
Why are glasswares should be sterilized in an
oven while culture media in an autoclave? ?