PEMBACAAN HASIL

MEDIA CAIR

Kediri, IIK Bhakti Wiyata

Novia Agustina, S.Si.,
M.Sc.
NaCl broth

• The medium was designed to aid in the differentiation of Enterococcus spp.

from streptococci by determining the ability of enterococci to grow in the presence
of 6.5% NaCl.
• Salt acts as a selective agent and interferes with membrane permeability and
osmotic equilibrium.
• Salt tolerant organisms will produce heavy growth in the broth and on solid agar
within 48 hours.
• Organisms that are capable of growing in the presence of a high salt
concentration will also ferment dextrose. Dextrose fermentation produces an acid
reaction which results in the bromcresol purple indicator turning yellow. Appearance
of a yellow color change in broth with indicator is indicative of a positive salt
tolerance test.
• Growth of organisms on high salt agar is also indicative of salt tolerance, but
the omission of bromcresol purple means no color reaction will occur.
• Organisms that do not grow on NaCl 6.5% agar are not salt tolerant. E.
faecalis, E. zymogenes, E. liquifaciens, and E. durans are among the Enterococcus
species that are salt tolerant.
INTERPRETATION OF RESULTS

• A positive salt tolerance test is indicated by growth and/or

turbidity in media without indicator. Media containing indicator will
produce a color change from purple to yellow. A negative test is
denoted by no growth and/or no color change. A negative test
indicates the organism is not capable of growing in a high salt
concentration.
PHYSICAL APPEARANCE

• NaCl 6.5% Media without Indicator should appear clear, and

light amber in color.
• NaCl 6.5% Media with Indicator should appear clear, and purple
in color.
Enterococcus faecalis growing in NaCl 6.5% Streptococcus pyogenes inhibited in NaCl 6.5%
Medium without Indicator. Incubated Medium without Indicator. Incubated aerobically for
aerobically for 24 hours at 35ºC 24 hours at 35ºC.
 Streptococci and Enterococci
Positive d.5% NaCI Growth
Negative

Oft€PO
Enterococcus faecalis growing in NaCl 6.5%
Medium with Indicator. Incubated aerobically for Streptococcus pyogenes inhibited in NaCl 6.5%
24 hours at 35ºC. Medium with Indicator. Incubated aerobically for 24
hours at 35ºC.
 Toleraoce Test

Uninoculated Negañve Positive

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).
• Beef extract is combined with peptone and sodium chloride to form
the basic bouillon. Yeast extract is added to provide vitamins and
minerals to help speed the growth of most organisms.
• Nutrient Broth can be enriched with other ingredients
such as carbohydrates, blood etc., for special purposes.
Quality control

Positive controls: Expected results

Staphylococcus aureus Turbid growth
Escherichia coli Turbid growth
Negative control:
Uninoculated medium No change

Bacillus subtilis produces
flocculent (flaky, clumped) growth
in liquid media;
Mycobacterium smegmatis growing on
the surface of the media, forming a
pellicle (film on the surface of the media
and and interior of the test tube, a
biofilm at the liquid / air interface).
Lactococcus lactis grows as a sediment at the Staphylococcus epidermidis growing in liquid
bottom of the tube; media creates a turbid, cloudy broth.
Pepton alkalis 1%

• a broth medium for the enrichment of Vibrio species from food,

water and clinical samples
• Alkaline Peptone Water is for the enrichment of Vibrio cholera
and Vibrio species from food, water and clinical samples. This
broth can also be used for direct microscopic examination of
samples using the hanging drop method.
• The 2% (w/v) sodium chloride incorporated in this medium
promotes the growth of Vibrio cholerae, while the alkalinity of
this medium inhibits most of the unwanted background flora.
Positive controls: Expected results

Vibrio parahaemolyticus Turbid growth

Vibrio vulnificus Turbid growth

Vibrio furnissii Turbid growth

Negative control:

Uninoculated medium No change

 Selenit broth

• Selenite broth is used as an enrichment medium for the isolation

of
Salmonella and Shigella from stool, urine, water and food products.
• It is the most widely used enrichment medium to isolate
Salmonella Typhi from fecal specimen of suspected patients
and/or from carriers during Salmonella outbreaks.
• Selenite is inhibitory to coliforms and certain other microbial
species found in faecal specimens.
• After the inoculation of the sample, selenite broth is incubated for
12- 18 hours at 35°C -37°C and then subculture is done in
selective agar (e.g., bismuth sulfite or desoxycholate citrate
agar).
• Casein enzymic hydrolysate provides nitrogenous substances
and carbon compounds required for bacterial growth.
• Lactose (fermentable carbohydrate) serves to maintain the pH
of medium. Any increase in pH will reduce the selective
activity of selenite. Proteus and Pseudomonas species which
are non-lactose fermenter appear to be resistant to effects of
selenite.
• Sodium phosphate maintains a stable pH and also lessens the
toxicity of selenite.
• Selenite exerts selective inhibitory effects. It is suggested that
it reacts with sulphur and sulphydral groups of in critical
cell components of microorganisms. Coliforms, fecal
streptococci, and Gram-positive organisms are inhibited by
sodium selenite.
Preparation of Selenite Broth:

• Suspend 23 g of the dehydrated powder in 1 litre of purified water (if twin

pack is available for use; dissolve 4g of sodium biselenite in 1 litre of
distilled water and then add 19g of Selenite Broth Base- check
manufacturer’s instruction labeled/available in the pack).
• Sterilise in a boiling water bath, or in free flowing steam, for 10 minutes.
Avoid overheating (as it is detrimental). Do not autoclave.
• Distribute in sterile test tubes. (Note: Discard the prepared medium if
large amount of red precipitate is seen at the bottom of the
tube/bottle)
• Label the side of each tube with date of preparation and batch number.
• Perform sterility testing.
• Store at 2-8°C
• Test samples of the finished product for performance using stable, typical
control cultures.
Fluid Selenite Cystine Medium
(Selenite Cystine Medium) (Twin Pach)
M025
1. Control
2. Salmonella Typhimurium ATCC 14028
3. Salmonella Choleraesuis ATCC
12011 4, Salmonella Typhi ATCC
6539
5. Eschefiehia coli ATCC 2592â
Oxgall
• Oxgall is a purified, dried form of ox bile used in bacteriological
media formulations for gastro-intestinal tract organisms, eg,
enterocossel agar.
• Bile is composed of fatty acids, bile acids, inorganic salts,
sulfates, bile pigments, cholesterol, mucin, lecithin, glycuronicacids,
porphyrins, and urea.
• The use of Oxbile insures a regular supply of bile, and
uniformity impossible to obtain with fresh materials.
• Oxbile is dehydrated fresh bile and prepared specifically for
differentiation of bile tolerant microorganisms. A 10% solution of
dehydrated bile is equivalent to a fresh bile solution.
• It is usually incorporated into media e.g., Bile Esculin Agar and
Brilliant Green Bile Agar, used for the determination of enteric
pathogens. Oxbile is also found in Littman Agar, a selective fungal
medium.
 Oxbile is used as a selective agent far the isolation
of Gram-negative microorganisms, inhibiting
Gram- positive bacteria. The major composition of
Oxbile is taurocholic and glycochalic acids.

Synonyms Bovine bile, Bacterial ogical ox bile

CAS No 8008-63-7

Bovine bile

Straw to beige or tan, free-flowing homogeneous

powder

Gold/greenish to amber, clear, co mplece

7.0—8.S

Loas on
Dzying

Total Bile
i50e

Storage and
Store powder at 4°C. Opened battles should
Seabilicy
be capped tightly and kept in a dark, low
humidity environment. Stable for 6 months
after receipt. Protect from moisture and lig ht
by keeping container tightly closed.
Cook meat

• Cooked Meat Medium is recommended for the cultivation of

aerobic, microaerophilic, and anaerobic microorganisms,
especially Clostridium species.
• Growth of spore-forming and non-spore-forming obligate
anaerobes is supported by this medium. Cooked Meat Medium
is also useful as an enrichment broth for cultivating organisms
from a very small inoculum
• Nutritional requirements needed by most bacteria are provided
by beef heart, peptone and dextrose.
• Dextrose, yeast extract, hemin and vitamin K are added to
enhance the growth of anaerobic microorganisms. Amino
acids and other nutrients are supplied by the muscle protein in
the heart tissue granules.
• Reducing substances, which permit the growth of strict
anaerobes, are supplied by the muscle tissue and the iron
filings.
• It is thought that the meat particles act as a reducing and
detoxifying substance, thereby disabling harmful by products
that may be produced by the replicating organism. Because
reducing substances are more available in denatured protein, the
meat particles are cooked before use in the medium.
LIMITATIONS

• Meat particles in the medium may cause turbidity, which

could be misinterpreted as positive growth.
• Meat particles blacken only in the presence of alkali, which is a
result of ammonia production by proteolytic enzymes.
• The reactions observed in the medium are useful for
characterization, not speciation, of the organism.
QUALITY CONTROL

Inoculatio Incubation
Test Organisms n Temperatur Atmospher Results
Method* Time
e e
Bacteroides fragilis
A 24-48hr 35°C Aerobic Growth

Streptococcus pyogenes
A 24-48hr 35°C Aerobic Growth

Clostridium perfringens
A 24-48hr 35°C Aerobic Growth
Bacteroides fragilis growing in
Cooked Meat Medium. Incubated
aerobically (with cap screwed down Uninoculated tube of Cooked Meat Medium
tightly) for 24 hours at 35ºC
Lactose Broth

• A liquid medium for use in the performance or confirmation

of the Presumptive Test for coliforms in water, milk, etc.
• These formulations can also be used to pre-enrich samples for
the recovery of Salmonella species. In processed foods,
Salmonella species can be present in low concentrations and
the cells may be found in a debilitated condition. Lactose
Broth provides an environment favorable for the recovery of
Salmonella.
• Tubes of Lactose Broth are inoculated with dilutions of the
samples and incubated at 35°C. Examination for gas formation is
carried out after 24 and 48 hours incubation. This presumptive
evidence of coliform organisms must be confirmed by further
tests.

• A pre-enrichment medium provides a higher ratio of

Salmonella to non-Salmonella bacteria after incubation. Most
non-Salmonella bacteria ferment lactose, while Salmonella does
not. When lactose- fermenting bacteria metabolize lactose in
the medium, the pH decreases, creating a bacteriostatic effect
on competing microorganisms.
 QUALITY CONTROL

Inoculation Incubation
Test Organisms Results
Method* Time Temperature Atmosphere
Escherichia coli Growth; acid and gas
A 18-48hr 35°C Aerobic
production**

Salmonella enterica Growth; no acid or

gas A 18-48hr 35°C Aerobic
production**
Quality control

Positive control: Expected results

Escherichia coli Turbid growth; acid and gas
production
Negative control:
Uninoculated medium No change
• Brillian Green Lactose Broth (BGLB)

• This medium is used to detect or confirm the presence of members of the coli-
aerogenes group; the brilliant green content suppresses anaerobic lactose
fermenters, such as Clostridium perfringens, and the medium is recommended for
the 44°C confirmatory test for Escherichia coli.
• The bile and brilliant green components inhibit the Gram-positive organisms, whilst
the coli-aerogenes group are recognised by the rapid formation of gas during
lactose fermentation.
• It is important that the inhibitory agents in the medium are balanced with the
nutrient and mineral components, so that Clostridium and Bacillus spores do
not give false positive reactions in the medium i.e. gas formation. Brilliant Green
Bile Broth is used in water, dairy and food analysis
Positive controls: Expected results

Escherichia coli Turbid growth; gas

Enterobacter aerogenes Turbid growth; gas

Negative control:
Staphylococcus aureus No growth