Negative stain:

• uses anionic dye to stain background

• useful for examining size and morphology of cells (No heat fixing)

Simple stain:

• uses one cationic dye to stain the cell

• useful for examining cell morphology and arrangement (heat fixing required)

Picture from General Microbiology Interactive Manual

Differential stain:

• uses two or more dyes to stain cells

• useful in differentiating cells based on certain properties (heat fixing required

o Gram Stain- differentiates Gram-positive cells from Gram-negative cells based on the amount of

peptidoglycan present in the cell wall

Primary dye: Crystal violet

Mordant: Grams Iodine

Decolorizer: 95% Ethanol

Secondary dye: Safranin

• Gram-positive- thick peptidoglycan layer retains dye causing the cell to stain purple

• Gram-negative- thin peptidoglycan layer easily decolorizes causing the cell to stain pink

Picture from https://ib.bioninja.com.au/options/untitled/b1-microbiology-organisms/gram-staining.html

o Acid-Fast Stain- differentiates acid-fast microbes, with high amounts of mycolic acids in their cell walls, and

non-acid-fast microbes with low amounts of mycolic acid in their cell walls

Primary dye: Carbol fuchsin

Steaming required as mycolic acid makes cells resistant to most dyes

Decolorizer: Acid Alcohol

Secondary dye: Methylene blue

• Acid-fast- carbol fuchsin is retained in the cell wall causing the cell to stain pink

o Mycobacterium tuberculosis

o Mycobacterium leprae

• Non-acid-fast- dye is easily decolorized, staining blue

Picture from https://www.thelabratartist.com/stains

Structural stain:

• Useful for examining certain characteristics of a call’s structure

o Endospore stain- allows you to identify the presence and location of spores (free spores or endospores)

Primary dye: Malachite green

Steaming required to penetrate spores

Decolorizer: water

Secondary dye: Safranin

• Vegetative cell- stains pink

• Endospore- stains green

Picture from http://faculty.ccbcmd.edu/courses/bio141/lecguide/unit1/prostruct/endobmmg.html

Selective Media:

• encourage or prevent the growth of certain bacteria

• contain inhibitors

o Phenylethyl Alcohol (PEA) Agar: useful for separating out Gram-positive bacteria from a mixture of bacteria

▪ Inhibitor: phenylethyl alcohol

• prevents the growth of Gram-negative by altering cell membrane permeability and

disrupting DNA synthesis

Picture from https://slideplayer.com/slide/14152438/

Differential Media:

• Distinguishes one type of microorganism from another

• contain indicators

o Motility Test: differentiates motile bacteria from nonmotile bacteria

▪ Indicator: Triphenyltetrazolium chloride (TTC)

• Nonmotile bacteria- red coloring confined to stab line

• Motile bacteria- red coloring spreads throughout the agar deep

Picture from https://microbenotes.com/motility-test-principle-procedure-and-results/

o Blood Agar (BA): differentiates bacteria based on hemolytic ability (useful for differentiating species of

streptococcus)

▪ Indicator: 5% sheep’s blood

• Beta (β) hemolysis: complete hemolysis of red blood cells

o Streptococcus agalactiae

o Streptococcus pyogenes

• Alpha (α) hemolysis: partial hemolysis of red blood cells

o Streptococcus pneumoniae

• Gamma (γ) hemolysis: no hemolysis of red blood cells

Picture from https://microbiologyinfo.com/haemolysis-of-streptococci-and-its-types-with-examples/

o Phenol Red Broth: enables us to determine if a bacteria is able to ferment a specific sugar (mannitol,

glucose and lactose)

▪ Indicator: Phenol red

• Control: red

• Sugar is fermented: acid is produced turning the broth yellow

o gas bubble trapped in Durham tube is also an indication of fermentation

• Sugar cannot be fermented: peptone deamination may be performed releasing NH3, raising

the pH of the broth and causing the broth to turn cerise

Picture from General Microbiology Interactive Manual

o Kliger’s Iron Agar (KIA): differentiates lactose fermenters, glucose fermenters, and sulfide production by

the enzyme cysteine desulfhydrase

▪ Indicator: phenol red- indicates fermenters from non-fermenters

▪ Indicator: ferric ammonium citrate- indicates sulfide production

• Glucose +, lactose +, cysteine desulfhydrase -:

o Mixed acid fermentation- tube turns completely yellow due to high acid

production, lifting and cracking may also be seen due to high gas production

o 2,3 butanediol fermentation- tube turns yellow due to acid production, but the tip

of the tube may revert back to a pink color as the products of this fermentation

pathway are not as stable

• Glucose +, lactose -, cysteine desulfhydrase -:

o bottom of tube turns yellow due to acid production

o top of the tube turns pink due to peptide deamination

• Glucose +, Lactose+, cysteine desulfhydrase +:

o Tube turns yellow and black

• Glucose +, Lactose-, cysteine desulfhydrase +:

o Tube turns pink and black (bottom of tube may remain yellow)

Picture from https://www.slideshare.net/Millatlaboratory/biochemical-tests-58986607

o MR-VP test: differentiates bacteria that ferment lactose using the mixed-acid pathway and the 2,3

butanediol

▪ Methyl red test: detects mixed-acid fermentation by analyzing pH

• Indicator: methyl red

o Positive for mixed-acid fermentation: high amount of acid produced by this

fermentation pathway overpower pH buffer and media will remain red

▪ E. coli

o Negative for mixed-acid fermentation: media will turn yellow/orange

▪ Enterobacter

▪ Voges–Proskauer (VP) test: detects 2,3 butanediol fermentation

• Indicator: Barret’s reagent (VP reagent A and VP reagent B)- detects acetoin, the precursor

molecule to 2,3-butanediol produced in the 2,3 butanediol fermentation pathway

o Positive for 2,3 butanediol fermentation: Barret’s reagent reacts with acetoin

forming a red band at the top of tube

▪ Enterobacter

o Negative for 2,3 butanediol fermentation: Barret’s reagent does not react with

acetoin and no red band formed

▪ E. coli

Pictures from http://w3.marietta.edu/~spilatrs/biol202/labresults/mrvp.html and https://microbeonline.com/voges-proskauer-test-principle-procedure-results/

o Tryptone Broth: differentiates bacteria that can and cannot produce the enzyme tryptophanase ▪ Enzyme: tryptophanase

• breaks down amino acid tryptophan into indole, pyruvate, and ammonia

▪ Indicator: Kovac’s reagent (DMABA)

• Tryptophanase positive: tryptophanase breaks down tryptophan producing

indole, indole reacts with DMABA forming a cerise color at the top of the media

o E.coli

• Tryptophanase negative: indole is not produced, orange color at the top of the

media

o Enterobacter

Picture from https://microbiologyinfo.com/indole-test-principle-reagents-procedure-result-interpretation-and-limitations/

o Nitrogen Reduction Test: differentiates bacteria that can and cannot produce nitrate reductase

▪ Enzyme: nitrate reductase

• Reduces nitrate (NO3) to nitrite (NO2)

▪ Indicator: sulfanilic acid (Nitrate I) and dimethyl-alpha-naphthylamine (Nitrate II)

• Nitrate I and II reacts with NO2 forming a red color

o Positive result: if nitrate reductase is produced NO3 is reduced to NO2, NO2, then

reacts with Nitrate I and II forming a red color

▪ if tube turns clear after the addition of Nitrate I and II iron is added

▪ Indicator: Zinc

• Reduces NO3 to NO2

o Positive result: tube remains clear as zinc does not react with NO3

o Negative result: tube turns red as zinc reacts with NO3

Picture from General Microbiology Interactive Manual

o Sulfide-Indole-Motility Medium (SIM):

▪ sulfide production- black precipitate formed (E and C)

• Salmonella

• Proteus

▪ Motility- cloudiness throughout the tube (E and C)

• Salmonella

• Proteus

▪ tryptophanase production- cerise color at the top of the tube (A and E)

Picture from https://www.pinterest.com/pin/97742254390320747

o DNase agar: differentiates staphylococcus species that can and cannot produce DNase

▪ Enzyme: DNase

• Cleaves the phosphodiester bond between DNA nucleotides

▪ Indicator: methyl green

• methyl green and DNA form a complex giving the agar a blue-green color

o DNase positive: if DNase is produced, a clearing will be shown around the bacterial

growth as DNase cleaves the DNA and breaks the complex with methyl green

▪ Staphylococcus aureus

o DNase negative: if DNase is not produced no change will occur

Picture from https://laboratoryinfo.com/deoxyribonuclease-dnase-test/

o Coagulase test: Rabbit plasma which contains fibrinogen is used to differentiate staphylococcus species

that can and cannot produce coagulase

▪ Enzyme: coagulase

• Converts fibrinogen to fibrin, which is visualized as clumping in the bottom of the tube

o Coagulase positive: clumping is seen

▪ Staphylococcus aureus

o Coagulase negative: clumping is not seen

Picture from http://www.uwyo.edu/molb2021/additional_info/summ_biochem/coagulase.html

o Catalase Test: differentiates bacteria that can and cannot produce catalase (useful for differentiating

between staphylococcus and streptococcus)

▪ Enzyme: superoxide dismutase (SOD) and Catalase

• SOD will catalyze the conversion of superoxide radicals into more H2O2

• Catalase will then convert H2O2 into water and oxygen

o Catalase positive: bubbles formed due to the production of O2 GAS

▪ Staphylococcus o Catalase negative: no bubbles formed

▪ Streptococcus

Picture from https://microbeonline.com/catalase-test-principle-uses-procedure-results/

o Urease Test: contains urea (useful for differentiating Proteus from other Gram-negative rods)

▪ Enzyme: Urease

• Removes NH3 from Urea

▪ Indicator: phenol red

• Urease positive: tube turns cerise as the release of NH3 raises the pH

o Proteus

• Urease negative: tube remains an orange color

o Salmonella

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

o Phenylalanine Slant: differentiates bacteria that can and cannot produce phenylalanine deaminase

▪ Enzyme: phenylalanine deaminase

• Converts phenylalanine into phenylpyruvic acid (PPA) and ammonia

▪ Indicator: ferric chloride (FeCl3)

• Phenylalanine deaminase positive: if PPA is produced FeCl3 reacts with PPA forming a

green color

• Phenylalanine deaminase negative: if PPA is not produced FeCl3 does not react

Picture from https://www.pinterest.com/pin/478648266621148624/

o Gelatin Hydrolysis Test: contains gelatin

▪ Exoenzyme: gelatinase

• Hydrolyzes gelatin

o Gelatinase positive: gelatin media is liquified

o Gelatinase negative: gelatin media remains solid

Picture from https://www.slideshare.net/HusseinAltameemi2/medical-microbiology-laboratory-clostridium-spp

o Oxidase Test:

▪ Enzyme: cytochrome c oxidase

• Oxidizes cytochrome c

▪ Indicator: dimethyl-p-phenylenediamine

• In the presence of dimethyl-p-phenylenediamine, cytochrome c oxidase adds electrons

back to cytochrome c and oxidizes dimethyl-p-phenylenediamine

o cytochrome c oxidase positive: dimethyl-p-phenylenediamine is oxidized turning

blue

o cytochrome c oxidase negative: cytochrome c oxidase is not produced and

dimethyl-p-phenylenediamine is not oxidized

Picture from https://quizlet.com/269962930/microbiology-bisc-260-lab-final-biochemical-tests-flash-cards/

o Lipase Test: spirit blue agar contains lipase reagent made of tributyrin (fat) and polysorbate 80 (emulsifier)

▪ Enzyme: lipase

• Hydrolyzes fats

▪ Indicator: Spirit Blue dye

• Lipase Positive: Bacteria that produce lipase will hydrolyze lipase reagent releasing spirt

blue dye and causing an intensified blue halo around the bacterial growth

• Lipase Negative: Bacteria that do not produce lipase do not hydrolyze lipase, therefore a

clearing will be shown around the bacterial growth

Picture from https://microbenotes.com/lipase-test-objectives-principle-procedures-and-results/

o Milk Agar: contains the protein casein which gives milk its milky white color

▪ Enzyme: caseinase

• Hydrolyzes casein into amino acids

o Caseinase positive: clear zone around the bacterial growth

o Caseinase negative: no clearing

Picture from General Microbiology Interactive Manual

o Litmus Milk: differentiates caseinase producing bacteria from non-caseinase producing bacteria and

lactose-fermenters from non-lactose-fermenters

▪ Enzyme: Caseinase

▪ Indicator: litmus

• Lactose fermentation +: litmus milk turns pink due to acid production

o Curding- excess acid production leads to denaturing of proteins in milk causing hard

curds

• Lactose fermentation -: If fermentation does not occur bacteria may perform peptone

deamination releasing NH3. This raises the pH of media causing the media turn blue (may

be limited to the top of tube as peptone deamination is oxygen dependent)

• Reduction: litmus milk only functions in an oxidized state, under reducing conditions the

milk loses its color and turns white (color will be limited to the top of the tube as the litmus

milk is exposed to oxygen and can be oxidized)

• Caseinase production +: caseinase breaks down the casein in litmus milk causing the milk

to turn clear

Picture from https://microbenotes.com/litmus-milk-medium-test/

o Starch Agar: contains starch

▪ Enzyme: amylase

• Hydrolyzes starch into glucose

o Amylase positive: When iodine is added to the plate it complexes with starch

creating a brown color. A clearing around the bacterial growth indicates no starch is

present, due to the production of amylase

o Amylase negative: no clearing shown after the addition of iodine

Picture from https://homepages.wmich.edu/~rossbach/bios312/LabProcedures/Starch%20Hydrolysis%20Medium.html

o Bacitracin Susceptibility Test: differentiates between β-hemolytic streptococcus species which are resistant

to sensitive to the antibiotic bacitracin

▪ Bacitracin resistant: no clearing around the bacitracin disk

▪ Bacitracin sensitive: clearing around the bacitracin disk

• Streptococcus pyogenes

Picture from https://microbeonline.com/bacitracin-test-principle-procedure-expected-results-and-quality-control/

Selective and Differential Media:

• Distinguishes one type of microorganism from another and encourages/prevents the growth of certain bacteria

• Contains both inhibitors and indicators

o MacConkey Agar (MAC): differentiates Gram-negative lactose-fermenters from Gram-negative non-

lactose-fermenters

▪ Inhibitor: Bile salts and crystal violet

• Prevents the growth of Gram-positive

▪ Indicator: neutral red

• Lactose fermenters: produce acid turning the media a reddish-pink color

o E. coli

o Enterobacter

• Non-lactose fermenters: show no color change

o Salmonella

o Shigella

o Proteus

Picture from https://cellularproducts1.files.wordpress.com/2012/01/97.jpg

o Eosin Methylene Blue (EMB) Agar: differentiates Gram-negative lactose-fermenters from Gram-

negative non-lactose-fermenters and identifies the pathway used for fermentation (mixed acid

fermentation or 2,3-butanediol fermentation)

▪ Inhibitor: Eosin Y and methylene blue

• Prevents the growth of Gram-positive

▪ Indicator: Eosin Y and methylene blue

• Mixed acid fermentation: large amount of acid is produced forming dark purple/black

or metallic green colonies

o E. coli

• 2,3-butanediol fermentation: less acid is produced forming light purple/pink colonies

o Enterobacter

• Non-lactose fermenters show no color change

o Salmonella

o Shigella

o Proteus

Picture from https://quizlet.com/250634614/mbio-3812-exam-3-flash-cards/

o Endo Agar (EA): differentiates Gram-negative lactose-fermenters from Gram-negative non-lactose-

fermenters and identifies the pathway used for fermentation (mixed acid fermentation or 2,3-

butanediol fermentation)

▪ Inhibitor: Sodium sulfite and basic fuchsin

• Prevents the growth of Gram-positive

▪ Indicator: basic fuchsin

• Mixed acid fermentation: large amount of acid is produced forming dark purple/black

or metallic green colonies

o E. coli

• 2,3-butanediol fermentation: less acid is produced forming light purple/pink colonies

o Enterobacter

o Klebsiella

• Non-lactose fermenters show no color change

o Salmonella

o Shigella

o Proteus

For a good visual refer to the Lab Manual

o Simmon’s Citrate Test: differentiates bacteria that can and cannot utilize citrate as a carbon source

(produce citrate permease and citrate lyase)

▪ Inhibitor: contains sodium citrate as the primary source of carbon, if bacteria cannot

breakdown citrate bacteria will not grow on media

▪ Indicator: bromothymol blue

• Positive result: bacteria that can breakdown citrate and utilize it as its primary source

of carbon will raise he pH of the media turning it blue

o Enterobacter aerogenes

• Negative result: bacteria that cannot breakdown and utilize citrate as its primary

source of carbon will not grow and will not change the pH of the media

o Escherichia coli

Picture from https://microbiologyinfo.com/citrate-utilization-test-principle-media-procedure-and-result/

o Salmonella-Shigella (SS) Agar: differentiates Gram-negative bacteria based on lactose fermentation

and sulfide production (useful for differentiating Salmonella and Shigella)

▪ Inhibitor: bile salts, brilliant green, and citrate

• Prevent the growth of Gram-positive and some Gram-negative (coliforms)

• Highly selective for Salmonella

▪ Indicator: neutral red- indicates lactose fermentation

• Lactose fermentation positive: colonies are pink or red

o E. coli (if they grow)

o Enterobacter (if they grow)

• Lactose fermentation negative: colonies are colorless

o Salmonella

o Shigella

o Proteus

▪ Indicator: sodium thiosulfate and ferric ammonium citrate-indicates sulfide production

• Sulfide production positive: forms a black center in the colony

o Salmonella

o Proteus

• Sulfide production negative: no black center

o Shigella

o Bismuth Sulfide Agar (BSA): differentials Gram-negative bacteria that can and cannot produce sulfide

▪ Inhibitor: bismuth sulfite and brilliant green

• Prevents the growth of Gram-positive and coliforms

• Highly selective for salmonella

▪ Indicator: ferrous sulfate

• Salmonella: sulfide production results in dark metallic colonies with metallic halo

• Enterobacter aerogenes: sulfide is not produced resulting in dark brown to green

colonies

• E. coli: does not grow well and produces clear to brown colonies

For a good visual refer to the Lab Manual

o Mannitol Salt Agar (MSA): differentiates between staphylococcus species that can ferment mannitol

and species that cannot ferment mannitol

▪ Inhibitor: 7.5% NaCl

• Prevents the growth of non-halotolerant

• Selects for the genus staphylococcus which are halotolerant

▪ Indicator: phenol red

• Positive result: staphylococcus species that can ferment mannitol produce acid causing

the tube to turn yellow

o Staphylococcus aureus

o Staphylococcus saprophyticus

• Negative result: staphylococcus species that cannot ferment mannitol cause the tube

to turn cerise

o Staphylococcus epidermis

Picture from https://superfarmer.wordpress.com/2008/05/26/mannitol-salt-agar-msa-test/