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## Actinomycete Isolation Agar

Actinomycetes are gram-positive bacteria, which show marked
chemical and morphological diversity but form a distinct evolutionary line of
organisms that range from coccoid and pleomorphic forms to branched filaments.
Actinomycetes form an integral part of soil, water and vegetation. Actinomycete
development leads to the formation of volatile metabolites. Traces of these
volatile metabolites are sufficient to impart disagreeable odor to water or a
muddy flavor to fish. Actinomycetes also cause disruptions in wastewater
treatment by forming massive growths, which are capable of producing thick foam
in the activated sludge process. Actinomyces Isolation Agar used for isolation
and propagation of Actinomycetes from soil and water was formulated by Olsen.

Actinomycete Isolation Agar contains sodium caseinate as
nitrogen source. Asparagine in addition to being an amino acid is also a source
of nitrogen. Sodium propionate is used as a substrate in anaerobic
fermentation. Dipotassium phosphate provides the buffering system. The
sulphates serve as source of Sulphur and metallic ions. Glycerol serves as an
additional source of carbon. Inoculate the plates with 1 drop of diluted
culture or specimen and spread over the surface using a sterile bent glass rod.
Incubate at 35-37°C for 40-72 hours. The media can be used for long term
storage after sufficient growth is obtained. Agar slants are used for
maintenance of cultures over a shorter period of time.

## Blood Agar

https://microbeonline.com/blood-agar-composition-preparation-uses-and-types-of-hemolysis/

Blood agar contains general nutrients and 5% sheep blood.  It is useful for cultivating fastidious organisms and for determining the hemolytic capabilities of an organism.

Some bacteria produce exoenzymes that lyse red blood cells and degrade hemoglobin; these are called hemolysins. Bacteria can produce different types of hemolysins.  Beta-hemolysin breaks down the red blood cells and hemoglobin completely. This leaves a clear zone around the bacterial growth. Such results are referred to as β-hemolysis (beta hemolysis). Alpha-hemolysin partially breaks down the red blood cells and leaves a greenish color behind.  This is referred to as α-hemolysis (alpha hemolysis).  The greenish color is caused by the presence of biliverdin, which is a by-product of the breakdown of hemoglobin.  If the organism does not produce hemolysins and does not break down the blood cells, no clearing will occur.  This is called γ-hemolysis (gamma hemolysis).

The hemolysins produced by streptococci perform better in an anaerobic environment.  Because of this, it is standard procedure to streak a blood plate and then stab the loop into the agar to provide an area of lower oxygen concentration where the streptolysins can more effectively break down the blood cells.

## Brain Heart Infusion

Brain heart infusion (BHI) is a growth medium for growing
microorganisms. It is a nutrient-rich medium, and can therefore be used to
culture a variety of fastidious organisms. In particular, it has been used to
culture streptococci, pneumococci and meningococcal, which can be otherwise
challenging to grow. BHI is made by combining an infusion from boiled bovine or
porcine heart and brain with a variety of other nutrients. BHI broth is often
used in food safety, water safety, and antibiotic sensitivity tests.

The earliest version of brain heart infusion media was made
in 1919 when Edward Rosenow combined dextrose broth with calf brain tissue to
grow streptococci. This was modified in 1923 by Russell Haden while working on
dental pathogens. Modern BHI typically uses an infusion from porcine brains and
hearts rather than calf brain tissue, and uses disodium phosphate as a buffer,
rather than the calcium carbonate used by Rosenow and Haden.

BHI typically contains infusion of beef or pig heart as well
as calf brain, a source of amino acids (often either digested gelatin or other
animal tissue), salt, disodium phosphate as a buffer, and glucose as a source
of sugar. Many formulations for BHI agar also exist, in which agar is added as
a gelling agent for growing plates of microorganisms.

Brain-Heart Infusion Broth can be supplemented with
antibiotics, varying amounts of sodium chloride, yeast extract, and serum to
provide a rich medium for bacteria, yeasts and pathogenic fungi.3 The addition
of 0.1% agar can be used to lower oxygen tension, providing an atmosphere to
support the growth of aerobic, microaerophilic, and obligate anaerobic
microorganisms.

## Brilliant Green Bile Broth

Brilliant Green Bile Broth 2% is one of the most widely used
medium for the detection of coliform bacteria in water, wastewater, foods, and milk
and dairy products. This medium is formulated as per APHA for the presumptive
identification and confirmation of coliform bacteria. This medium is also
recommended by the ISO Committee for enumeration of coliforms by most probable
number technique. Peptone serves as a source of essential nutrients. Lactose is
the fermentable carbohydrate. *Bile*
inhibits gram-positive bacteria whereas the gram-negative bacteria are
inhibited by brilliant green*. Production of gas from lactose fermentation*
is detected by incorporating inverted Durham's tube, which indicates the
positive evidence of fecal coliform since non fecal coliforms growing in this
medium do not produce gas. Further gas production in EC broth (M127) at 45°C
used as a confirmation of fecal coliform. Gram-positive spore formers may
produce gas if the bile or brilliant green inhibition is weakened by reaction
with food material. During examination of water samples, growth from
presumptive positive tubes showing gas in Lactose Broth (M026) or Lauryl Tryptose
Broth (M080) is inoculated in Brilliant Green Bile Broth 2% (M121). Gas
formation within 48 ± 2 hours confirms the presumptive test).

Brilliant Green Bile Broth 2% is used for the detection of
coliform bacteria in water, food, and dairy products in a laboratory setting.
Brilliant Green Bile Broth 2% is not intended for use in the diagnosis of
disease or other conditions in humans.

The coliform group of bacteria includes aerobic and
facultative anaerobic, Gram-negative, non-spore-forming bacilli that ferment
lactose and form acid and gas at 35°C within 48 hours. Members of the
Enterobacteriacae comprise the majority of this group, but organisms such as
Aeromonas spp. may also be included. Procedures to detect and confirm coliforms
are used in testing water, foods, dairy products and other materials. Brilliant
Green Bile Broth 2% is used to confirm a positive presumptive test result.

The formulation for Brilliant Green Bile Broth was developed
by the Association of Official Analytical Communities (AOAC) and the American
Public Health Association (APHA). The basal medium is composed of peptone,
which contains 2% bile, and brilliant green dye. Bile is inhibitory to
gram-positive microorganisms, while brilliant green dye inhibits selected
gram-negative bacilli. Lactose-fermenting organisms resistant to these
inhibitors are detected by the production of gas. Gas production is noted by
the appearance of bubbles in the Durham tube.

## Lactose Broth

Examination of water, foods, ingredients and raw materials,
for the presence of marker groups such as coliforms is one of the most common
tests in a microbiology laboratory, partly because of the relative ease and
speed with which these tests can be accomplished. Where it is claimed that
drinking water has been processed for safety, the finding of such organism
demonstrates a failure of the process. It is a valuable bacterial indicator for
determining the extent of fecal contamination of recreational surface waters or
drinking water. Lactose Broth is recommended by APHA in the performance and
confirmation of the presumptive test for coliform bacteria in water, food and
milk. This medium was initially listed as an alternative to Lauryl Sulfate
Broth in the presumptive Standard Total Coliform Multiple-Tube (MPN) Test for
water analysis. Although it is not the original formulation, Lactose Broth
provides excellent results in Eijkman Assays of gas production at 45°C, which
is a characteristic of Escherichia coli. While preparing this medium it is
important to avoid overheating and to distribute it into tubes before
sterilization. Peptic digest of animal tissue and beef extract in the medium
supply essential nutrients to the organisms. Lactose is a fermentable
carbohydrate for the coliforms. Tubes of Lactose Broth are inoculated with
dilutions of water or milk, etc. under test, and incubated at 35°C and examined
for gas formation after 24 and 48 hours. Members of the coliform group are
defined as aerobic and facultative anaerobic gram-negative and non-sporing
bacilli, which ferment lactose with gas formation within 48 hours at 35°C. In
testing dairy products, Lactose Broth is used only in the completed test (3).
Large water samples may require double strength Lactose Broth to minimize the
final volume

## LB Broth

Lysogeny broth (LB), a nutritionally rich medium, is
primarily used for the growth of bacteria. The initialization is also commonly,
albeit incorrectly, taken to mean Luria broth, Lennox broth, or Luria-Bertani
medium. According to its creator Giuseppe Bertani, the abbreviation LB was
actually intended to stand for lysogeny broth.[1] The formula of the LB medium
was published in 1951 in the first paper of Bertani on lysogeny. In this
article he described the modified single-burst experiment and the isolation of
the phages P1, P2, and P3. He had developed the LB medium to optimize Shigella
growth and plaque formation.

LB media formulations have been an industry standard for the
cultivation of Escherichia coli as far back as the 1950s. These media have been
widely used in molecular microbiology applications for the preparation of
plasmid DNA and recombinant proteins. It continues to be one of the most common
media used for maintaining and cultivating laboratory recombinant strains of
Escherichia coli.[8] For physiological studies however, the use of LB medium is
to be discouraged: Hiroshi Nikaido describes the limitations of peptides and
amino acids as nutrients (with major switches in early log phase) and limited
availability of Mg and Ca in this important commentary, available online.

There are several common formulations of LB. Although they
are different, they generally share a somewhat similar composition of
ingredients used to promote growth, including the following:

* Peptides and casein peptones
* Vitamins (including B vitamins)
* Trace elements (e.g. nitrogen, sulfur, magnesium)
* Minerals

Sodium ions for transport and osmotic balance are provided
by sodium chloride. Tryptone is used to provide essential amino acids such as
peptides and peptones to the growing bacteria, while the yeast extract is used
to provide a plethora of organic compounds helpful for bacterial growth. These
compounds include vitamins and certain trace elements.

## MacConkey Agar

**Selective**: It contains bile salts (to inhibit most Gram-positive bacteria), crystal violet dye (which also inhibits certain Gram-positive bacteria)

**Differential**: neutral red dye (which turns pink if the microbes are fermenting lactose).

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. The crystal violet and bile salts inhibit the growth of gram-positive organisms which allows for the selection and isolation of gram-negative bacteria. Enteric bacteria that have the ability to ferment lactose can be detected using the carbohydrate lactose, and the pH indicator neutral red.

Composition:

* Peptone – 17 g
* Proteose peptone – 3 g
* Lactose – 10 g
* Bile salts – 1.5 g
* Sodium chloride – 5 g
* Neutral red – 0.03 g
* Crystal violet – 0.001 g
* Agar – 13.5 g
* Water – add to make 1 litre; adjust pH to 7.1

+/− 0.2

## Mannitol Salt Agar

A.              B.                C.                D.

A. Gram + *Staphylococcus*: fermenting mannitol: medium
turns yellow (e.g. *S. aureus*)

B.Gram + *Staphylococcus*: not fermenting mannitol, medium does not change
color (e.g. *S. epidermidis*)

C.Gram + *Streptococcus*: inhibited growth

D. Gram -: inhibited growth

Selective: 7.5% NaCl for selectivity of halotolerant
species of *Staphylococcus*

Differential: Phenol Red pH
Mannitol fermentation vs. non-fermenting.

Mannitol salt agar or MSA is a commonly used selective and
differential growth medium in microbiology. It encourages the growth of a group
of certain bacteria while inhibiting the growth of others. This medium is
important in medical laboratories as one method of distinguishing pathogenic
microbes in a short period of time. It contains a high concentration (about
7.5%-10%) of salt (NaCl), making it selective for Gram-positive bacteria
(Staphylococcus and Micrococcaceae) since this level of salt is inhibitory to
most other bacteria It is also a differential medium for mannitol-fermenting
staphylococci, containing carbohydrate mannitol and the indicator phenol red, a
pH indicator for detecting acid produced by mannitol-fermenting staphylococci.
Staphylococcus aureus produces yellow colonies with yellow zones, whereas other
coagulase-negative staphylococci produce small pink or red colonies with no
color change to the medium. If an organism can ferment mannitol, an acidic
byproduct is formed that causes the phenol red in the agar to turn yellow. It
is used for the selective isolation of presumptive pathogenic (pp)
Staphylococcus species.

## Nitrate Reduction

https://microbiologyinfo.com/nitrate-reduction-test/

## MR-VP Broth: See Biochemical Tests for MR-VP

https://microbiologynote.com/methyl-red-and-voges-proskauer-test-principle-result/

Methyl Red (MR) and Voges-Proskauer (VP) broth is used as a part of the IMViC tests as the medium in which both the Methyl Red and Voges-Prosakuer tests can be performed. It is a simple broth that contains peptone, buffers, and dextrose or glucose.

Different bacteria convert dextrose and glucose to pyruvate using different metabolic pathways. Some of these pathways produce unstable acidic products which quickly convert to neutral compounds. Some organisms use the butylene glycol pathway, which produces neutral end products, including acetoin and 2,3-butanediol. Other organisms use the mixed acid pathway, which produces acidic end products such as lactic, acetic, and formic acid. These acidic end products are stable and will remain acidic.

The Methyl Red test involves adding the pH indicator methyl red to an inoculated tube of MR-VP broth. If the organism uses the mixed acid fermentation pathway and produces stable acidic end-products, the acids will overcome the buffers in the medium and produce an acidic environment in the medium. When methyl red is added, if acidic end products are present, the methyl red will stay red.

NOTE: **Methyl** red differs from **Phenol** red (which is used in the fermentation test and the MSA plates) in that it is yellow at pH 6.2 and above and red at pH 4.4 and below. Phenol red turns yellow below a pH of 6.8. If you get these two pH indicators confused, you will have a difficult time interpreting test results.

The VP test detects organisms that utilize the butylene glycol pathway and produce acetoin. When the VP reagents are added to MR-VP broth that has been inoculated with an organism that uses the butylene glycol pathway, the acetoin end product is oxidized in the presence of potassium hydroxide (KOH) to diacetyl. Creatine is also present in the reagent as a catalyst. Diacetyl then reacts to produce a red color. Therefore, red is a positive result. If, after the reagents have been added, a copper color is present, the result is negative.

The MR and VP tests are particularly useful in the identification of the *Enterobacteriaceae*.

**Important:**

Instead of making you aliquot inoculated broth into sterile test tubes, we have already divided the broth. Inoculate two MR-VP broths for each organism you wish to test. For the lab exercise, you would inoculate two MR-VP broths with *Escherichia coli* and two MR-VP broths with *Enterobacter cloacae*. After incubation, you would perform an MR test on one tube of *Escherichia coli* and one tube of *Enterobacter cloacae*, and you would perform a VP test on the other tube of *Escherichia coli* and the other tube of *Enterobacter cloacae*.

## Nutrient Gelatin

https://microbiologynote.com/gelatin-hydrolysis-test-purpose-principle-procedure-result/

### Gelatin hydrolysis test Principle

The gelatin proteins derived from the animal connective tissue, collagen. Gelatin is produced when collagen is boiled in water. A gelatin hydrolysis test is used to detects the presence of gelatinases. Gelatinases are extracellularly secreted by some bacteria which hydrolyze or digest gelatin.

The hydrolysis/digest process of gelatin is takes place in two sequential reactions. In the first reaction, gelatinases degrade gelatin to polypeptides. Then in the second step, the polypeptides are further converted into amino acids.

For the detection of Gelatin hydrolysis, a nutrient media is used known as gelatin medium. This gelatin medium is a mixture of peptic digest of animal tissue (peptone), beef extract, and gelatin. The gelatine helps to solidify the medium and as well as functions as a substrate for gelatinase activity. When nutrient gelatin tubes are stab-inoculated with a gelatinase-positive bacterium, the secreted gelatinases will hydrolyze the gelatin resulting in the liquefaction of the medium.  Since gelatin is digested and is no longer able to gel, the medium will remain liquid when placed inside a refrigerator or in an ice bath.

A nutrient gelatin medium inoculated with a gelatinase-negative bacterium will remain solid after the cold treatment. The medium can be inoculated with both aerobic and anaerobic bacteria and incubated as appropriate.

### Requirement

* Nutrient Gelatin Media
* Mercuric chloride (HgCl2)
* Inoculating needle
* Incubator at 37°C
* Pipettes

### Nutrient gelatin stab method

All the methods used for the detection of gelatinase production, all contain gelatin as the substrate. The nutrient gelatin stab method is the standard and most commonly employed method. This method gives easy and accurate results (medium liquefaction) and reduces the use of reagents or chemicals that may be dangerous to health.

1. Stab-inoculate the inoculum of an 18- to 24-hour-old test bacteria into the tubes containing nutrient gelatin.
2. Incubate the tubes along with the control tube at 25°C, or at the test bacterium’s optimal growth temperature, for up to 1 week, and check everyday for gelatin liquefaction.
3. Gelatin usually liquefies at 28°C and above, so to endorse that liquefaction was due to gelatinase activity, the tubes are dipped in an ice bath for 15 to 30 minutes.
4. Afterward, tubes are tilted to observe if gelatin has been hydrolyzed.
5. The hydrolyzed gelatin will result in a liquid medium even after exposure to cold temperature (ice bath), while the uninoculated control medium will remain solid.
6. For weak positive results, incubate the inoculated nutrient gelatin tube longer until complete liquefaction is observed. The hydrolysis of gelatin indicates the secretion of gelatinase by the test organism into the medium.
7. Note that some bacterial species, e.g., *Proteus vulgaris*, may require up to 14 days of incubation to yield a positive result.
8. Aseptic techniques and appropriate biosafety protocols should always be strictly observed and practiced in the microbiology laboratory.

Some gelatinase-positive organisms in nutrient gelatin stab method can produce the enzyme at a slower rate. Thus, the following tips are recommended:

* Stab-inoculate nutrient gelatin tubes with a heavy inoculum of the test bacteria.
* Incubate nutrient gelatin tubes for a longer period (7 days up to 14 days).
* Use smaller test tubes for the nutrient gelatin medium (13 by 100 mm).
* Use a smaller volume of nutrient gelatin (2 to 3 ml) in the tubes.

## Phenyl Ethanol Agar

https://www.austincc.edu/microbugz/phenyethyl\_alcohol\_agar.php

Phenylethyl alcohol agar (PEA) is a selective medium used to cultivate Gram positive organisms. The active ingredient, phenylethyl alcohol, inhibits or markedly reduces growth of Gram negative organisms by interfering with DNA synthesis. PEA also prevents *Proteus* species from swarming across the surface of the agar.

*Staphylococcus aureus*, a Gram positive organism, grows on this PEA plate while *Serratia marcescens*, a Gram negative organism, does not.

Paper: Antibacterial activity of phenethyl alcohol and resulting membrane alterations

## Phenol Red Broth

Phenol Red Broth
is a general-purpose differential test medium typically used to differentiate
gram negative enteric bacteria.  It contains peptone, phenol red (a pH
indicator), a Durham tube, and one carbohydrate.

We use
three different kinds of phenol red broths. One contains glucose; one contains
lactose, and the last contains sucrose.  The objective of the exercise is
to determine which organisms can utilize each sugar.

Phenol red
is a pH indicator which turns yellow below a pH of 6.8 and fuchsia above a pH
of 7.4. If the organism is able to utilize the carbohydrate, an acid by-product
is created, which turns the media yellow.  If the organism is unable to
utilize the carbohydrate but does use the peptone, the by-product is ammonia,
which raises the pH of the media and turns it fuchsia.

When the
organism is able to use the carbohydrate, a gas by-product may be produced. If
it is, an air bubble will be trapped inside the Durham tube.  If the
organism is unable to utilize the carbohydrate, gas will not be produced, and
no air bubble will be formed.

More
in-depth information on the biochemical pathways involved in carbohydrate
fermentation can be found in your lab manual.

A.    B. C.  D. No pic

A.  The tube
inoculated with *Escherichia
coli*produces acid and gas. It would be read as A/G.

B.  The tube
inoculated with *Enterococcus
faecalis*, produces acid but no gas. It would be read as A/-.

C.  The *Micrococcus luteus*provides
no reaction and would be read as -/-.

D.  The *Alcaligenes faecalis*degrades
peptone and creates an alkaline by-product.  It would be read as K.

## Simmon's Citrate Agar

https://microbiologyinfo.com/simmons-citrate-agar-composition-principle-uses-preparation-and-result-interpretation/

### Simmons Citrate Agar- Composition, Principle, Uses, Preparation and Result Interpretation

Last updated: August 15, 2019 by Sagar Aryal

**Simmons Citrate Agar** is an agar medium used for the differentiation of Enterobacteriaceae based on the utilization of citrate as the sole source of carbon.

In the early 1920s, **Koser** developed a liquid medium formulation for the differentiation of fecal coliforms from the coliform group. **Simmons** later modified this formulation to produce a solid medium that eliminated potential errors when interpreting growth.

### Principle of Simmons Citrate Agar

Simmons Citrate agar is used to test an organism’s ability to utilize citrate as a source of energy. **Ammonium Dihydrogen Phosphate** is the sole source of **nitrogen**. **Dipotassium Phosphate** acts as a **buffer**. **Sodium Chloride** maintains the **osmotic balance** of the medium. **Sodium Citrate** is the sole source of **carbon** in this medium. **Magnesium Sulfate** is a cofactor for a variety of metabolic reactions. **Bacteriological agar** is the solidifying agent. Organisms capable of utilizing ammonium dihydrogen phosphate and citrate will grow unrestricted on this medium. If citrate can be used, the microbe will accumulate alkaline/basic byproducts.

Bacteria that can grow on this medium produce an enzyme, **citrate-permease**, capable of converting **citrate** to **pyruvate**. **Pyruvate** can then enter the organism’s metabolic cycle for the production of **energy**. Growth is indicative of utilization of citrate, an intermediate metabolite in the **Krebs cycle**.

When the bacteria metabolize **citrate**, the **ammonium salts** are broken down to **ammonia**, which increases **alkalinity**. The shift in pH turns the **bromthymol blue** indicator in the medium from **green to blue** above pH 7.6.

### Uses of Simmons Citrate Agar

1. It is used for the differentiation of Gram-negative bacteria on the basis of citrate utilization.
2. Simmons Citrate Agar may be used to differentiate citrate-positive *Salmonella enteritidis* and members of *Salmonella* subgenus II, III and IV from the citrate-negative *Salmonella typhi, Salmonella paratyphi* A, *Salmonella pullorum* and *Salmonella gallinarum*.
3. Simmons Citrate Agar is primarily used to aid in the identification of Enterobacteriaceae. Uses include:

* *Escherichia coli* (usually -) and *Shigella* spp. (-) from other commonly encountered
* Enterobacteriaceae (variable +).
* *Edwardsiella* spp. (-) from *Salmonella* spp. (usually +)
* *Serratia proteamaculans* (+) from *Yersinia pseudotuberculosis* (-) and *Yersinia enterocolitica*
* (usually -)
* *Klebsiella*-Enterobacter groups (usually +) from *E. coli* (usually -)
* *Proteus rettgeri* (+) from *Morganella morganii* biogroups 1 and 2 (-)
* *Yokenella regensburgei* (+) from *Hafnia alvei* (-)
* *Leminorella grimontii* (+) from *L. richardii* (-)
* *Acidovorax delafieldii* (+) from *A. facilis* (-) and  *A. temperans* (-)

### Result Interpretation on Simmons Citrate Agar

**Positive growth** (i.e. citrate utilisation) produces an alkaline reaction and changes the colour of the medium from green to bright blue.

**Examples:** *Serratia* and the majority of the *Enterobacter, Citrobacter, Klebsiella, Proteus* and *Providencia* species, except *Morganella morganii* and *Klebsiella rhinoscleromatis*.

**Negative test** (i.e. no citrate utilisation) the colour of the medium remains unchanged.

**Examples:** *Escherichia coli,* *Shigella, Yersinia, Edwardsiella* species, etc.

### Limitations of Simmons Citrate Agar

1. It is important not to carry over any nutrients into the citrate medium because this will result in false positive tests.
2. Some organisms are capable of growth on citrate and do not produce a color change. Growth is considered a positive citrate utilization test, even in the absence of a color change.
3. Tests with equivocal results should be repeated.
4. The reactions of this medium alone are not sufﬁcient for identiﬁcation to the species level.
5. The inoculum should be from a pure, overnight culture grown on a solid medium and not from a broth suspension.

### Quality Control on Simmons Citrate Agar

**Positive control:** *Klebsiella pneumoniae* ATCC® 13883. Growth; colour change of medium to blue

**Negative control:** *Escherichia coli* ATCC® 25922. Inhibited or no growth; no colour change of medium

### References

1. Thermo Fisher Scientific Inc., Dehydrated Culture Media- SIMMONS CITRATE AGAR.
2. Sisco Research Laboratories Pvt. Ltd.- Simmon’s Citrate Agar (59437).
3. Austin Community College.
4. Hardy Diagnostics- SIMMONS CITRATE AGAR (L80).
5. Intuitive Systems, Inc.- Simmons’ Citrate Agar Slant.
6. Scharlab- Simmons Citrate Agar (01-177).
7. Merck Microbiology Manual 12th Edition- SIMMONS Citrate Agar.
8. Dalynn Biologicals- SIMMONS CITRATE AGAR (TS62).
9. Remel- Citrate Agar (Simmons).
10. Conda Lab- SIMMONS CITRATE AGAR ISO 10273 (1014).
11. Sigma-Aldrich, Inc.- SIMMONS CITRATE AGAR (S3681).
12. HiMedia Laboratories Pvt. Ltd.- Simmons Citrate Agar (M099).
13. Acumedia Manufacturers, Inc.- SIMMONS CITRATE AGAR (7156).

## Triple Sugar Iron

https://microbiologyinfo.com/triple-sugar-iron-tsi-test/

## Urease Test

https://microbiologyinfo.com/urease-test-principle-media-procedure-and-result/