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Culture Media:

A culture medium is a liquid or gel designed to support the growth of microorganisms or cells.
Culture media provide the appropriate biochemical and biophysical environment. A large variety
and types of culture media have been developed with different purposes and uses. Culture media
are employed in the isolation and maintenance of pure cultures of bacteria and are also used for
identification of bacteria according to their biochemical and physiological properties. There are
different types of media for growing different types of cells.

Type:

The most common growth media for microorganisms are nutrient broths; liquid media are often
mixed with agar and poured via sterile media dispenser into Petri dishes to solidify. These agar
plates provide a solid medium on which microbes may be cultured.

Nutrient media

Nutrient media contain all the elements that most bacteria need for growth and are non-selective,
so they are used for the general cultivation and maintenance of bacteria kept in laboratory culture
collection. An undefined medium (also known as a basal or complex medium) is a medium that
contains:

a carbon source such as glucose for bacterial growth


water
various salts needed for bacterial growth
a source of amino acids and nitrogen (e.g., beef, yeast extract)
This is an undefined medium because the amino acid source contains a variety of
compounds with the exact composition being unknown.

A defined medium (also known as chemically defined medium or synthetic medium) is a


medium in which

all the chemicals used are known


no yeast, animal or plant tissue is present
Some examples of nutrient media include:

Nutrient agar
Trypticase soy agar
Mueller-Hinton agar.
Selective media
Selective media allows the growth of certain type of organisms, while inhibiting the growth of
other organisms. This selectivity is achieved in For example, if a microorganism is resistant to a
certain antibiotic, such as ampicillin or tetracycline, then that antibiotic can be added to the
medium in order to prevent other cells, which do not possess the resistance, from growing.
Like-wise, the selective inhibition of some types of microorganisms can be studied by adding
certain dyes, antibiotics, salts or specific inhibitors that will affect the metabolism or enzymatic
systems of the organisms. For example, MacConkey agar for gram negative bacteria, Bacitracin
chocolate agar for Haemophillus influenzae.

Differential media
Differential media or indicator media distinguish one microorganism type from another growing
on the same media. This type of media uses the biochemical characteristics of a microorganism
growing in the presence of specific nutrients or indicators (such as neutral, phenol red, eosin y,
or methylene blue) added to the medium to visibly indicate the defining characteristics of a
microorganism. This type of media is used for the detection of microorganisms and by molecular
biologists to detect recombinant strains of bacteria.
Examples of differential media include:

Blood agar (used in strep tests), which contains bovine heart blood that becomes transparent
in the presence of hemolytic Streptococcus
Eosin methylene blue (EMB), which is differential for lactose fermentation
MacConkey agar (MCA), which is differential for lactose fermentation
Mannitol salt agar (MSA), which is differential for mannitol fermentation

Transport media
Transport media should fulfill the following criteria:

Temporary storage of specimens being transported to the laboratory for cultivation.


Maintain the viability of all organisms in the specimen without altering their concentration.
Contain only buffers and salt.
Lack of carbon, nitrogen, and organic growth factors so as to prevent microbial
multiplication.
Transport media used in the isolation of anaerobes must be free of molecular oxygen.
Examples of transport media include:

Thioglycolate broth for strict anaerobes.


STGG medium (Skim Milk Tryptone Glycerol medium )
Certain bacterial inhibitors- for gonococci, and buffered glycerol saline for enteric bacilli.
Venkataraman Ramakrishna(VR) medium for V. cholerae.

Enriched media
Enriched media contain the nutrients required to support the growth of a wide variety of
organisms, including some of the more fastidious ones. They are commonly used to harvest as
many different types of microbes as are present in the specimen. Blood agar is an enriched
medium in which nutritionally rich whole blood supplements the basic nutrients. Chocolate
agar is enriched with heat-treated blood (4045 C), which turns brown and gives the medium
the color for which it is named.

Media preparation

Two types of media need to be prepared.

1) Culture media for the isolation of bacterial pathogen and,

2) Biochemical media for the identification of the isolate.

Consumables & Reagents:


Distilled water: Distilled water is the major component of media. Good quality distilled water
has pH level at 7.
Container: Conical flask is suitable container for the preparation of media. Size of container
should be selected according to the required volume of the media. At least one-third (1/3) of the
volume of the flask should remain empty when filled with the media.
Gloves: Gloves should be used to avoid contamination.
Petri dishes: Sterile Petri dish (90 x 15 mm) should be used that retain maximum of 20 ml
culture media.
Tubes: Sterile tubes (10 ml, 16 x 160 mm) should be used for the preparation of biochemical
media.
Dehydrated media: In preparation of complex culture media it is advisable to use ready-made
standardized dehydrated media to ensure good performance and reproducibility. It is less
expensive than individual chemical constituents.
Blood collection kit: Dehydrated agar products require 5-7%defibrinated blood to prepare blood
agar media and Mueller-Hinton blood agar. We will use 5% sheep blood to prepare the culture
media supplemented with blood.
Capital equipment:
Water bath: Water bath must be filled with distilled water up to safe level. Temperature should
be set as required for the media preparation.
Autoclave machine: Dissolved media should be autoclaved at 121C for 20 minutes. Before
autoclaving, the flask containing dissolved media should be closed tightly with rounded cotton at
the tip (Photograph). This will facilitate air passing out of the flask at high temperature.
Laminar flow: Autoclaved media should be poured into the plate inside the laminar flow to
prevent air contamination. Bubble produced on the plate after pouring will be destroyed by
giving a heat on it.

Common precautions:
1. Media preparation should be done aseptically in the closed separate room.
2. Differences in pH of the water may changes the quality of media.
3. Dehydrated media are hygroscopic and absorb water. When exposed to moisture, it rapidly
becomes unfit for use. So it should be kept in a cool dry place.
4. Weigh the media rapidly and capped tightly as soon as possible after removing the
approximate amount. Do not return small amount into the stock bottle.
a. The pH of the dehydrated medium should not require adjustment. If required, minor
adjustments should be carried out using 0.1 mol/l (N/10) sodium hydroxide when the
medium is too acid, and 0.1 mol/l (N/10) hydrochloric acid when too alkaline.

Culture media preparation

Culture media for the isolation of blood and CSF specimen will be prepared at the central lab.
Each batch of media will go through a process of quality assurance (QA) during preparation and
quality control (QC) after then. Media will be distributed among the site labs only if the batch of
prepared media has passed quality control test. The procedure for QC test has described at
section 8.2.7
Chocolate agar (CA) plate
Principle
Chocolate agar (CA) plate is a boiled blood agar
plate, in which red blood cells have been lysed by
heating very slowly at 80C. These facilitate to
extract two growth factor, NAD and hematin inside
from erythrocytes. Haemophilusinfluenzae can
grow on only chocolate agar plate due to
availability of these two growth factors in the Fig: Chocolate Agar plate
media. Plate does not chocolate; it is named
chocolate agar only for its color.

Procedure:
1. Suspend the dehydrated powder (Oxoid CM0055, Hampshire, England) with required
amount of distilled water according to manufacturers instruction. In case of Oxoid product
CM0055, suspend 40g of dehydrated media in 1 liter of distilled water.
2. Mix thoroughly. Heat with frequent agitation and boil for 1 minute to completely dissolve the
powder.
3. Measure the pH of the media. It should be within the range of 7.2-7.6 at room temperature.
Adjust the pH if required.
4. Insert a dry, tight cotton plug on the top of the flask.
5. Autoclave the media at 121C for 15 minutes.
6. Transfer the autoclaved media to a 50C water bath.
7. When the agar has cooled to 50C, aseptically add 5% sterile sheep blood and mix gently.
8. Heat the liquid media mixed blood at 80C for 15 minutes to lyse the blood. The red color of
the media will turn into chocolate.
9. Dispense 20 ml onto a sterile petri dish inside the laminar flow. Remove the bubbles from the
plate if formed after dispensing.
10. Place the plate inside the leveled surface of the laminar flow and keep at room temperature to
solidify the media.
11. Label the plate with media name, date and batch number.
12. Send a randomly selected media for QA test. Store the rest at 2-8C in sealed plastic bags to
prevent the loss of moisture.

Bacitracin chocolate agar: By adding 6 ml Bacitracin Solution with 600 ml of Chocolate agar
Bacitracin chocolate agar for selective growth of H. influenzae is prepared.
Blood Agar (BA) Plate

Principle
Blood agar (BA) plate containing 5% sheep blood
is a differential growth medium that differentiates
pathogenic bacteria based on the interaction of
sheep's blood, bacterial hemolytic enzymes
(appendix2) and morphology of the colonies. The
pH of media maintained at 7.3 stabilizes red blood
corpuscles and favors the formation of a clear
Fig: Blood Agar plate
haemolysis zone.

Procedure:
1. Suspend the dehydrated powder (Oxoid CM0055, Hampshire, England) with required
amount of distilled water according to manufacturers instruction. In case of Oxoid product
CM0055, suspend 40g of dehydrated media in 1 litre of distilled water.
2. Mix thoroughly. Heat with frequent agitation and boil for 1 minute to completely dissolve the
powder.
3. Measure the pH of the media. It should be within the range of 7.2-7.6 at room temperature.
Adjust the pH if required using 0.1 mol/l (N/10) sodium hydroxide when the medium is too
acid, and 0.1 mol/l (N/10) hydrochloric acid when too alkaline.
4. Insert a dry, tight cotton plug on the top of the flask.
5. Autoclave the media at 121C for 15 minutes.
6. Transfer the autoclaved media to a 50C water bath.
7. When the agar has cooled to 50C, aseptically add 5% sterile sheep blood and mix gently.
During mixing, avoid forming air bubbles.
8. Dispense 20 ml onto a sterile petri dish inside the laminar flow cabinet. Remove the bubbles
from the plate if formed after dispensing.
9. Place the plate inside the leveled surface of the laminar flow and keep at room temperature to
solidify the media.
10. Label the plate with media name, date and batch number.
11. Send a randomly selected media for QA test. Store the rest at 2-8C in sealed plastic bags to
prevent the loss of moisture.

As most of Streptococcus pneumoniae are gentamicin resistant, Gentamicin Blood agar is used
for selective growth of S. pneumoniae. Adding 600 L of Gentamycin Solution with 600 ml of
Blood agar Gentamycin Blood agar is prepared.
MacConkey Agar Plate (MAC)

Principle
MacConkey agar is a selective media for most of the Gram-
negative bacteria except H. influenzae and N. meningitidis; it
can also differentiate the gram negative bacteria based on
lactose fermentation. MacConkey agar media contains bile
salts that inhibit most of the Gram-positive bacteria, except
Enterococcus and some species of Staphylococcus. It also
contains Crystal violet dye which inhibits certain Gram-
positive bacteria and neutral red dye that stains lactose
fermenting microbes. By utilizing the lactose available in the
medium, Lactose producing bacteria (E. coli, Enterobacter
andKlebsiella) produce acid, which lowers the pH of the agar Fig: MacConkey Agar plate
below 6.8 and results in the appearance of red/pink colonies.
Non-Lactose fermenting bacteria (Serratia and Citrobacter)
use peptone, forms ammonia, which raises the pH of the agar
and leads to the formation of white or colourless colonies.

Preparation:
1. Suspend the dehydrated powder (Oxoid CM0115, Hampshire, England) with required
amount of distilled water according to manufacturers instruction.
2. In case of Oxoid product CM0115, suspend 51.5 gram of dehydrated media in 1 litre of
distilled water.
3. Mix thoroughly. Heat with frequent agitation and boil for 1 minute to completely dissolve the
powder.
4. Measure the pH of the media. It should be within the range of 7.2-7.6 at room temperature.
Adjust the pH if required as described above.
5. Insert a dry, tight cotton plug on the top of the flask.
6. Autoclave the media at 121C at 15 PSI for 15 minutes.
7. Transfer the autoclaved media to a 50C water bath.
8. When the agar has cooled to 50C, dispense 20 ml onto a sterile petri dish inside the laminar
flow cabinet. Remove the bubbles from the plate if formed after dispensing.
9. Place the plate inside the leveled surface of the laminar flow and keep at room temperature to
solidify the media.
10. Label the plate with media name, date and batch number.
11. Send a randomly selected media for QA test. Store the rest at 2-8C in sealed plastic bags to
prevent the loss of moisture.

Mueller-Hinton Agar Plate (MA):


Principle
Mueller Hinton Media contains beef infusion and casamino
acids, as a source of energy and nutrients. It also contains
starch, acts as a colloid that provide protection against toxic
material in the medium. The levels of tetracycline and
sulfonamide inhibitors, thymidine, thymine, magnesium and
calcium ions are controlled so as not to interfere with
susceptibility testing (disk diffusion and E-strips) and to yield
good growth. Susceptibility testing is done using this media of
those organisms, which do not require blood for their growth. Fig: Mueller-Hinton Agar plate
E.g.- Klebsiella sp., Salmonella sp., E.coli, Acinetobacter,
Pseudomonas
Preparation:
1) Suspend the dehydrated powder (Oxoid CM0337, Hampshire, England) with required amount
of distilled water according to manufacturers instruction. In case of Oxoid product CM0337,
suspend 38 g of dehydrated media in 1 liter of distilled water.
2) Mix thoroughly. Heat with frequent agitation, and boil for 1 minute to completely dissolve the
powder.
3) Measure the pH of the media. It should be within the range of 7.2-7.6 at room temperature.
Adjust the pH if required.
4) Insert a dry, tight cotton plug on the top of the flask.
5) Autoclave the media at 121C for 15 minutes.
6) Transfer the autoclaved media to a 50C water bath.
7) When the agar has cooled to 50C, dispense 20 ml onto a sterile petri dish. Remove the
bubbles from the plate if formed after dispensing.
8) Place the plate inside the leveled surface of the laminar flow and keep at room temperature to
solidify the media.
9) Label the plate with media name, date and batch number.
10) Send a randomly selected media for QA test. Store the rest at 2-8C in sealed plastic bags to
prevent the loss of moisture.

Mueller-Hinton Blood Agar Plate (MB)

Principle
Mueller Hinton Media contains beef infusion and casamino acids, as a source of energy and
nutrients. It also contains starch, acts as a colloid that provide protection against toxic
material in the medium. The levels of tetracycline and sulfonamide inhibitors, thymidine,
thymine, magnesium and calcium ions are controlled so as not to interfere with susceptibility
testing (disk diffusion and E-strips) and to yield good growth. Susceptibility testing is done
using this media of those organisms, which do not require blood for their growth. E.g.-
Streptococcus sp., Staphylococcus sp.,

Preparation:
1. Suspend the dehydrated powder (Oxoid CM0337, Hampshire, England) with required
amount of distilled water according to manufacturers instruction. In case of Oxoid product
CM0337, suspend 38 g of dehydrated media in 1 litre of distilled water.
2. Mix thoroughly. Heat with frequent agitation and boil for 1 minute to completely dissolve the
powder.
3. Measure the pH of the media. It should be within the range of 7.2-7.6 at room temperature.
Adjust the pH if required.
4. Insert a dry, tight cotton plug on the top of the flask.
5. Autoclave the media at 121C for 15 minutes.
6. Transfer the autoclaved media to a 50C water bath.
7. When the agar has cooled to 50C, aseptically add 5% sterile sheep blood and mix gently.
During mixing, avoid forming air bubbles.
8. Dispense 20 ml onto a sterile petri dish inside the laminar flow. Remove the bubbles from the
plate if formed after dispensing.
9. Place the plate inside the leveled surface of the laminar flow and keep at room temperature to
solidify the media.
10. Label the plate with media name, date and batch number.
11. Send a randomly selected media for QA test. Store the rest at 2-8C in sealed plastic bags to
prevent the loss of moisture.
Trypticase soy agar
is an antibiotic mixture of vancomycin, colistin and nystatin which is incorporated
460l into 100ml MB plate to permit selective isolation of Neisseria meningitidis.
Salmonella-Shigella Agar:

Principle:

SS Agar is a differential, selective medium for the


isolation of Shigella and Salmonella species from
pathological specimens, suspected foodstuffs, etc.
Gram-positive and coliform organisms are
inhibited by the action of the selective inhibitory
components brilliant green, bile salts, thiosulphate
and
VCN citrate.

Thiosulphate
Brain Heart in combination
Infusion Brothwith iron also acts as
(BHI)
an indicator for sulphide production, which is
indicated
STGG by blackening in the centres of the
colonies.
Fig: SS Agar Media

Preparation:

Suspend the dehydrated powder (Oxoid CM0099, Hampshire, England) with required amount of
distilled water according to manufacturers instruction. In case of Oxoid product CM0337,
suspend 63 g of dehydrated media in 1 litre of sterile distilled water.
12. Mix thoroughly. Heat with frequent agitation and boil for one minute until complete
disolution. Do not autoclave. Cool to 45 50C and distribute in Petri dishes. Measure the
pH of the media. It should be within the range of 7.2-7.6 at room temperature. Adjust the pH
if required.
13. Insert a dry, tight cotton plug on the top of the flask.
14. Autoclave the media at 121C for 15 minutes.
15. Transfer the autoclaved media to a 50C water bath.
16. Dispense 20 ml onto a sterile petri dish inside the laminar flow. Remove the bubbles from the
plate if formed after dispensing.
17. Place the plate inside the leveled surface of the laminar flow and keep at room temperature to
solidify the media.
18. Label the plate with media name, date and batch number.
19. Send a randomly selected media for QA test. Store the rest at 2-8C in sealed plastic bags to
prevent the loss of moisture.
Skim Milk Tryptone Glycerol Glucose (STGG) medium

Procedure

1. Dissolve glycerin (30 ml) in 300 ml distilled water, mix well and autoclave 121C for 15
minutes. Next cool down to room temperature.
2. Add TSB (9 gm) and skim milk (6 gm), mix well. .
3. Autoclave for 10 min at 121C.
4. After media have cooled, dispense 1ml volumes of the broth into 1.5 ml screw capped
tubes.
5. Store at 4C for up to 6 months, or indefinitely if frozen at ULT.

Triptic Soy Broth (TSB)

Procedure

1. Dissolve 30 g in 1 liter distilled water, mix well and distribute into final containers.
2. Autoclave for 10 min at 121C.
3. Store at 4C for.

Brain Heart Infusion Broth (BHI)

Procedure

1. Dissolve 37 g in 1 liter distilled water, mix well and distribute into final containers.
2. Autoclave for 10 min at 121C.
3. Store at 4C for.

Todd-hewitt broth

Procedure

1. Dissolve 36.4 g in 1 liter distilled water, mix well and distribute into final containers.
2. Autoclave for 10 min at 121C.
3. Store at 4C for.

Biochemical test media preparation

Bio-chemical media is required for the primary identification of isolates. In our lab, we use 3
types of biochemical media for most Gram negative clinical isolates. Further biochemical tests
and serological typing must be performed for definite identification and confirmation of
organisms.
Each batch of prepared media will go through a process of quality assurance (QA) and quality
control (QC). Media will be distributed among the site labs only if the batch of prepared media
has passed quality control test. The procedure for QC test has described in this section earlier.

Triple Sugar Iron Agar (TSI).

Principle
A composite medium for the differentiation of Enterobacteriaceae according
to their ability to ferment lactose, sucrose and glucose, and to produce
hydrogen sulphide. Its sucrose content permits the recognition and exclusion
of sucrose-fermenting species. These organisms may ferment lactose slowly
or not at all during the incubation period, but they attack sucrose readily.
Phenol red, pH indicator is used in response to the acid produced during the
fermentation of these sugars. The combination of ferric ammonium citrate
and sodium thiosulfate enables the detection of hydrogen sulfide (H2S)
production.

Preparation
1. Suspend the dehydrated powder (Oxoid CM0277, Hampshire, England)
of TSI with required amount of distilled water according to
manufacturers instruction. In case of Oxoid product CM0277, suspend
13 gm of dehydrated media in 200 ml of distilled water.
2. Mix thoroughly. Heat with frequent agitation and boil for 1 minute to
completely dissolve the powder.
3. Measure the pH of the media. It should be within the range of 6.8-7.2 at
room temperature. Adjust the pH if required. Fig: Kligler Iron Agar
4. Dispense 7 ml in each large size screw cap tubes (approx. 16160 mm)
before doing autoclave.
5. Loosen the cap of tubes containing media and autoclave at 121C for 15
minutes.
6. Allow the medium to solidify in a sloped position to give a butt 25-30
mm deep and slope 20-25 mm long (the butt should be longer than the
slope).
7. Label the plate with media name, date and batch number.
8. Send a randomly selected media for QC test. Store the rest at 2-8C in a
dark place.
Note Hydrogen sulfide production may be evident on KIA but negative on
TSI agar in few cases. Studies by Bulmash and Fulton (ref.-3, 1964) showed
that the utilization of sucrose could suppress the enzymatic mechanisms
responsible for H2S production. Padron and Dockstader (ref.-4, 1972) found
that not all H2S -positive Salmonella are positive on TSI.

MIU (Motility Indole Urease) Agar


Principle: MIU media contain urea and phenol red to detect urease
enzyme produced by organism. This media is a semi-solid media that
confirm the motility of the organism by changing the transparent
appearance of the media into fuzzy.

Fig:MIUAgar

Preparation:
1. Suspend the dehydrated powder (HIMEDIA M1076, Mumbai, India) with required amount
of distilled water according to manufacturers instruction. In case of HIMEDIA product
M1076, suspend 3.6 gm of dehydrated media in 190 ml of distilled water.
2. Mix thoroughly. Heat with frequent agitation and boil for 1 minute to completely dissolve the
powder.
3. Measure the pH of the media. It should be within the range of 6.6-7.0 at room temperature.
Adjust the pH if required.
4. Insert a dry, tight cotton plug on the top of the flask.
5. Autoclave the media at 121C for 15 minutes.
6. Transfer the autoclaved media to a 50C water bath.
7. When the agar has cooled to 50C, aseptically add 10 ml of sterile 40% urea solution in 190
ml media and mix gently. During mixing, avoid forming air bubbles.
8. Dispense 5 ml in each tube (approx. 16150 mm).
9. Allow the media to solidify Place the tube in upright position and do not make any slope in
this media. Cover with cap or cotton at the top.
10. Label the plate with media name, date and batch number.
11. Send a randomly selected media for QC test. Store the rest at 2-8C.
Simmons Citrate Agar (SCA)

Principle
Simmons citrate agar media allows the growth of the organism which
can exclusively depend on citrate as a sole source of energy.
Bromophenol blue change of color of the medium if citrate is utilized.

Fig: Simmons Citrate


Agar

1. Preparation: Suspend the dehydrated powder (Oxoid CM0155, Hampshire, England) with
required amount of distilled water according to manufacturers instruction. In case of Oxoid
product CM0155, suspend 4.6 gm of dehydrated media in 200 ml of distilled water.
2. Mix thoroughly. Heat with frequent agitation and boil for 1 minute to completely dissolve the
powder.
3. Measure the pH of the media. It should be within the range of 6.8-7.2 at room temperature.
Adjust the pH if required.
4. Dispense 7 ml in each large tube (approx. 16150 mm) before doing autoclave.
5. Loose the cap of the tube containing media and autoclave at 121C for 15 minutes.
6. Allow the medium to solidify in an oblique position to prepare only slant (no butt is
required).
7. Label the plate with media name, date and batch number.
8. Send a randomly selected media for QC test. Store the rest at 2-8C.

QA/QC of the Culture media

pH and color

1. Examine the pH of the media before autoclave.


2. Adjust pH if required. Examine the color of media and take
a decision for its accuracy and examine any crystal Quadrant
formations or variations in color (for agars).
3. The containers of media should be thoroughly examined
for cracks or defects, and all defective units should be
discarded.
Sterility testing
1. Perform sterility test on all media prepared in the laboratory.
2. Incubate one plate from each batch, at 37C for 48 hours and
3. Incubate again on each successive 7 days until the supply in the refrigerator is depleted or the
expiry date is reached.
4. If expected results are not attained, the QA technologist must be informed.

Quality Control of solid media:


1. Use fresh ATCC culture (no later the 24 hours old culture) of S. aureus, E. coli, S.
pneumoniae and H. influenzae.
2. Dry one media from each batch prepared.
3. Mark a quadrant on the backside of each plate.
4. Touch single colony from the above mentioned cultures and inoculate them in separate
quadrant. Incubate at 37C for 24 hours with 5% CO2.
5. Media will be tagged as QC pass, if the media yield the following growth.

Table 1: Chart of the QC result


Media for Batch pH S. E. S. H. Results
QC no. aureus coli pneumoniae influezae
Blood agar 2010- 7.2- X Pass
### 7.6
Chocolate 2010- 7.2- Pass
agar ### 7.6
MacConkey 2010- 7.2- X X X Pass
agar ### 7.6
Mueller- 2010- 7.2- X Pass
Hinton ### 7.6
blood agar
Mueller- 2010- 7.2- X X Pass
Hinton ### 7.6
agar

QA/QC of the Biochemical media

Bio-chemical media used to identify the pathogens needs to prepare aseptically. Each batch of
media should go through a process of quality control.
Inoculate all three media with freshly prepared culture of Salmonella typhi, Proteus and
Klebsiella.
Observe the reaction of each media and declare as Passed if they give the following reaction.

Strain Simmo Motility Indole Urease Triple Sugar Iron Results


ns (MIU) (TSI)
Citrate Motili Indol Urea Sla But H2S Gas
ty e nt t
Klebsiel +89 -11 - + +78 -22 A A - + Pass
laoxytoc
a
Proteus +57 -43 + - + K A + + Pass
mirabili
s
S. typhi - + - - K A + d Pass

Quantitative QC of Broth:

On day one, pick isolated colony (single colony for S. aureus, E. coli, H.influenzae & 5 colonies
of S. pneumonia) and dissolve in normal saline for serial dilution up to 10-5. Inoculate 100l of
last two dilutions (10-4& 10-5) of each organism in TSB broth (containing sheep blood) and
simultaneously, subculture on respect media. Incubate at 37C for 18hrs with 5% CO2.

On day two, Count the colonies on the plate and subculture the incubated broth on media as
described earlier.

On day three, correlate the colony counts between day 1 and day 2 to ensure the efficiency of
broth. Growth will obviously appear on the plate from day2 of respective dilutions, if at least one
colony appeared on the plate from day 1. Otherwise, the broth should be quarantined for its
efficiency.

QC of antibiotic disk

The current use and accuracy of any susceptibility test system is dependent on good quality
control practices. The Clinical and Laboratory Standards Institute (CLSI), (Performance
Standards for Antimicrobial Susceptibility Testing; Twenty-Fifth Informational Supplement)
provide guidelines for the selection of QC strains and zone diameter of different antibiotics by
disk diffusion method.
Table 2: QC results.

Normal zone range Normal zone range


Antibiotic name
(ATCC: E.coli) (ATCC: S.aureus)
Amikacin 19-26
Ampicillin 16-22
Azithromycin 21-26
Carbenicillin 23-29
Cefepime 31-37
Cefixime 23-27
Ceftazidime 27-35
Ceftriaxone 29-35
Cephalexin
Choramphenicol 21-26
Ciprofloxacin 30-40
Clindamycin 24-30
Cotrimoxazole 23-29
Doxicycline 18-24
Erythromycin 22-30
Gentamycin 19-26
Imipenem 26-32
Levofloxacin 29-37
Linezolid 25-32
Meropenem 28-34
Moxifloxacin 28-35
Nalidaxic Acid 22-28
Netilmycin 22-30
Nitrofurantoin 20-25
Ofloxacin 29-33
Oxacillin
Piperacillin 24-30
Tetracyclin 18-25
Tobramycin 18-26
Vancomycin 17-21
Optochin (ATCC:
SPN)=19
XV Factor (+/-)
Appendix 1: Media composition

1.1 Blood agar

Formula

Lab lemco powder 10.0g/L

Peptone 10 g/L

Nacl 5 g/L

Agar 15 g/L

pH 7.3 0.2 at 25 0 C

1.2 Chocolate Agar

Formula

Lab lemco powder 10.0 g/L

Peptone 10 g/L

Nacl 5 g/L

Agar 15 g/L

pH 7.3 0.2 at 25 0 C

1.3 MacConkey Agar

Fromula:

Peptone 20 g/L

Lactose 10 g/L

Bile salts on. 31.5

NaCl 5.0 g/L

Neutral red 0.03 g/L

Agar 15 g/L
pH 7.3 0.2 at 25 0 C

1.4 Mueller Hinton Blood Agar

Formula:

Beef, dehydrated infusion from 3.0 g/L

Casein hydrolysate 17.5 g/L

Starch 1.5 g/L

Agar 17 g/L

pH 7.3 0.2 at 25 0 C (5% sheep blood added)

1.5 KliglerIron Agar (KIA-Oxoid)

Formula:

Lab-lemco powder 3 g/L

Yeast extracts 3 g/L

Peptone 20 g/L

Sodium chloride 5 g/L

Lactose 10 g/L

Glucose 1 g/L

Ferric citrate 0.3 g/L

Sodium thiosulphate 0.3 g/L

Phenol red 0.05 g/L

Agar 12 g/L

pH 7.422 at 25C

1.6 MIU Medium Base (Hi-media)


Formula:

Casein enzymic hydrolysate 10 g/L

Dextrose 1 g/L

Sodium chloride 5 g/L

Phenol red 0.01 g/L

Agar 2 g/L

pH 6.80.2 at 25C

1.7 Simmons citrate agar (oxoid)

Formula:

Magnesium sulphate 0.2 g/L

Ammonium dihydrogen phosphate 0.2 g/L

Sodium ammonium phosphate 0.8 g/L

Sodium citrate, tri basic 2 g/L

Sodium chloride 5 g/L

Bromothiamol blue 0.08 g/L

Agar 15 g/L

pH 7 2 at 25C

1.8 Salmonella shigella agar (SS AGAR)

Formula:

Peptone 5.0 g/L

Lactose 5.0 g/L

Bile salts 10 g/L


Sodium citrate 8.5 g/L

Sodium thiosulphate 10 g/L

Ferric citrate 1.0 g/L

Brilliant green 0.00033 g/L

Neutral red 0.025 g/L

Agar 15g/L

pH 7.00.2 at 25C

1.9 Triptic Soy Broth

Formula

Pancreatic digest of casein 17 g/L

Enzymatic digest of soya bean 3.0 g/L

Sodium chloride 5.0 g/L

Di-potassium hydrogen phosphate 2.5 g/L

Glucose 2.5 g/L

pH 7.00.2 at 25C

1.10 Brian Heart Infusion Broth (BHI)

Formula

Brain infusion solids 12.5 g/L

Beef heart infusion solids 5.0 g/L

Proteose peptone 10.0 g/L

Sodium chloride 5.0 g/L

Di-sodium phosphate 2.5 g/L


Glucose 2.0 g/L

pH 7.40.2 at 25C

1.11 Todd-hewitt broth

Formula

Infusion from 450 g fat-free minced meat 10.00 g/L

Tryptone 20.0 g/L

Sodium chloride 2.0 g/L

Sodium bicarbonate 2.0 g/L

Disodium phosphate 0.4 g/L

Glucose 2.0 g/L

pH 7.80.2 at 25C

1.12 Skim Milk Tryptone Glycerol Glucose (STGG) medium

Formula:

Skim milk powder 2.0 g

Ttryptone soy broth powder 3g

Glucose 0.5g

Glycerol 10ml

Distilled water 100ml

1.13 Selective broth preparation for S. pneumoniae:

Formula:

TSB 9.5ml

Lysed Blood 0.5mL

Gentamycin 9.5L
Appendix 2: Reagent

2.1 Saline (0.85% NaCl)

Dissolve the sodium chloride in distilled water in a flask or bottle, using a magnetic stirrer.
Distribute 1 ml into each of screw cap tubes. Label as 0.85% NaCl with an expiration date of 1
year. Sterilize by autoclaving at 121 C for 15 min.

2.2 10X TBE buffer

Reagent Amount (g/L)

Tris base 108 (g/L)

Boric acid 55 (g/L)

EDTA (0.5m) 40ml/L

Adjust the PH at 8.0

2.3 10X TE Buffer

100 mMTris-cl (PH 7.4)


10 mM EDTA (PH 8.0)

2.4 2% sodium deoxycholate

Materials

2 g sodium deoxycholate
20 ml sterile distilled water

2.5 1X Phosphate Buffered Saline (PBS Buffer)

8g of NaCl
0.2g of KCl
1.44g of Na2HPO4
0.24g of KH2PO4
800ml sterile distilled H2O.

Other reagent preparation?


QA/QC of site laboratory(Naziat), QC from WHO(t we do for QC, Take suggestion from
Hafiz Vai )wh and from CDC (from Hasan).