LAB EXAM

DIFFERENTIAL TESTS

• These tests were chosen because they provide a sampling of metabolic diversity available for testing

• Categories

  • energy metabolism (fermentation and respiration)

  • utilization of a medium component

  • decarboxylation and deamination of amino acids

  • hydrolytic reactions

  • multiple reactions performed from a single combination medium

  • antimicrobial susceptibility

  • Miscellaneous

COLONY MORPHOLOGY AND COLOR

• Pigmentation/Color

  • can often point to a specific species

• Shape/Margins

  • can help narrow down the category

GRAM STAIN

• main purpose is to categorize bacteria as either Gram-positive or Gram-negative

• Main stain: crystal violet

• Mordant: Gram’s Iodine

• Secondary or counterstain: Safranin

• Gram-Positive

  • purple

  • thicker peptidoglycan

  • Staphylococcus aereus

• Gram-Negative

  • pink

  • membrane is more porous

  • Klebsiella Pneumoniae

ENDOSPORE STAIN

• main purpose is distinguished dormant, highly resistant endospores from vegitative cells

• helps narrow down bacterium based on if it is a spore former or not

• Primary stain: Malachite Green

• Counterstain: Safranin

• Spore formers

  • Clostridium genera

  • Bacillus genera

ACID FAST STAIN (DID NOT DO)

• to identfiy bacterial that have mycolic acid in their walls

  • thick waxy lipid rich cell wall

• for Mycobacterium

  • tuberculae

  • leprae

• Primary stain: Carbol Fuschin

• Wash: Acid Alcohol

• Secondary or counterstain: Methyl Blue

FLUID THIOGLYCOLLATE MEDIA (OXYGEN REQUIREMENTS)

• FTM tube

• main purpose is to test the aerotolerance of bacteria

• establishes and oxygen gradient within a single tube

• Obligate aerobes

  • only grow at the top

  • Pseudomonas aeruginosa

• Obligate anaerobes

  • only grow at the bottom

  • Clostridium tetani

• Facultative anaerobes

  • grow throughout the tube but are densest at the top

  • Klebsiella Pneumoniae

• Microaerophiles

  • form a distinct, thin band just below the top layer

  • Haemophilus influenzae

• Aerotolerant anaerobes

  • grow evenly throughout entire tube

  • Lactobacillus rhamnosus

MOTILITY (STAB CULTURE)

• main purpose to identify if bacterium is capable of self-directed movement or not

• use a reduced agar concentration so that the bacteria can swim outward if capable

  • standard agar is too solid for them to be able to move

• Positive

  • cloudy growth that flares outward from central stab line

  • tube may be cloudy throughout

  • Proteus vulgaris

• Negative

  • distinct initial stab line with no outwards growth

  • surrounding agar is colorless

  • Klebsiella pneumoniae

GELATINASE (STAB CULTURE)

• Main purpose is to detect the presence of gelatinases in the bacterium

  • if they are present, bacterium can hydrolyze gelatin

    • the cell can absorb individual amino acids and use them for metabolic purposes

• Gelatin is a protein derived from collagen

• Differs from other solid media because gelatin is also a substrate for enzymatic activity

• Positive

  • secreted gelatinases will liquify the medium

  • staphylococcus aureus

• Negative

  • medium remains solidified

  • Klebsiella pneumoniae

*gelatin liquifies at 28 degress celsius, therefore has to be stored at 25 degrees celsius

CARBOHYDRATE FERMENTATION

consisted of inverted Durham tube and the specfied carbohydrate & shift in pH levels

• detects acid production (via color change) and gas production (via bubbles in inverted Durham tube)

• if fermenter, can break down the sugar and convert it to energy without the use of oxygen

  • pH becomes more acidic

• GLUCOSE

  • Positive

    • Acid production: broth turns yellow

    • Gas production: bubbles

  • Negative

    • Red

    • Pseudomonas aeruginosa

• LACTOSE

  • Positive

    • Acid production: broth turns yellow

    • Gas production: bubbles

  • Negative

    • Red

    • Pseudomonas aeruginosa

• MANNITOL

  • Positive

    • Acid production: broth turns yellow

    • Gas production: bubbles

  • Negative

    • Red

    • Pseudomonas Aeruginoa

*Klebsiella pneumoniae positve for all

METHYL RED/VOGUES-PROSKAUER

uses MVP broth that contains peptone, glucose, and a phosphate buffer

• if positive, pH is more acidic

• METHYL RED

  • purpose is to identify is a bacterium is capable of performing a mixed acid fermentation

  • Positive

    • red color indicates a positive result, suggesting the production of stable mixed acids during fermentation.

    • E coli

  • Negative

    • yellow is negative

    • orange is inconclusive

    • K. Pneumoniae

• VOGUES-PROSKAUER

  • purpose is to identify organisms that are able to ferment glucose and produce acetoin

  • adding VP reagents oxidizes acetoin to diacetyl, which reacts with guanidine nuclei to form a red color

    • Positive

      • red color

      • K. pneumoniae

    • Negative

      • no color change or development of a copper color

      • E. coli

NITRATE REDUCTION

uses an inverted Durham tube and potassium nitrate broth to detect if an organism is producing nitrate reductase which allows them to use nitrate as a terminal electron acceptor during anaeroic respiration

• many gram negative bacteria have nitrate reductase and perform single step reduction of nitrate to nitrite

• Denitirification: converts nitrate to molecular nitrogen or nitrous oxide

  • if not observed visually, sulfanilic acid is added to test for nitrate to nitrite reduction

• Positive

  • denitrification: if the Durham tube contains gas

    • Pseudomonas aeruginosa

  • singe step reduction: red color

    • K. pneumoniae

• Negative

• denitrification: if no gas in Durham tube is observed

• single step reduction: no color change

• Staphylococcus species

**powdered zinc can be used to further test if the bacterium is positive or negative if none of the above work

CITRATE UTILIZATION

Uses Simmons citrate Agar slant to assess the organism's ability to utilize citrate as its sole carbon source and perform citrate fermentation

• provides the means for bacterium that possess citrate permease to transport citrate and perform fermentation

  • must also be able to survive with ammonium

• Positive

  • results in a color change from green to blue of the agar slant

    • in the absence of color to do incompleted uncubation, growth on media also indicates positive result

  • bacterium hydrolyzed citrate into oxaloacetate and acetate using citrate lyse, which was then converted to pyruvate

  • bacterium can convert ammonium phosphate to ammonia and ammonium hydroxide

  • K. pnemumoniae

• Negative

  • agar slant stays green

  • E. coli

OXIDASE PRODUCTION

Oxidase reagent is added to see if the reducing agent becomes oxidized in the presence of bacteria, indicating that the bacteria can produce cytochrome c oxidase

• Positive

  • color change to blue occurs within seconds

  • Pseudomonas aeruginosa

• Negative

  • no color change occurs

  • if you wait too long, most will change blue eventually

  • K. pneumoniae

CATALASE PRODUCTION

uses hydrogen peroxide to observe if the enzymes catalse and superoxide dismutase is present, which breaks down hydrogen peroxide

• Positive

  • oxygen gas bubbles form, indicating the bacterium is breaking down the hydrogen peroxide

  • K. pneumoniae

• Negative

  • no oxygen gas bubbles form

  • obligate anaerobes always test negative

  • Clostridium

HYDROLYSIS REACTIONS

Agars used to determine if the bacterium produce an enzyme that can break down a given material

• SPIRIT BLUE AGAR (LIPID)

  • Positive    

    • bacteria produces lipase

    • clear zones appear around the growthindicating the breakdown of lipids in the medium, which is a characteristic sign of lipolytic activity.

    • P. aeruginosa

  • Negative

    • bacteria does not break down lipids

    • there may still be growth, but no clear zoneswill be observed, indicating that the bacteria do not possess lipolytic enzymes.

    • K. pneumonia

• MILK AGAR (CASEIN)

  • Positive

    • bacterium diffuses into medium and creates a zone of clearing where the casein has been hydrolyzed

    • bacterium produces casease

    • K. pneumonia

  • Negative

    • may still be growth, but no clear zone around growth

    • E. coli

• STARCH AGAR (STARCH)

  • Positive

    • when Iodine is added, there is a clearing observed around the organism

    • organism makes alpha amylase, beta amylase, and glucosidase

    • Clostridium

  • Negative

    • when Iodine is added, no zone of clearing around the organism

    • K. pneumoniae

INDOLE PRODUCTION

Determines if the bacterium produces tryptophanase to break down tryptophan into pyruvate, ammonia, and indole

• after incubation, Kovac’s reagent is added, which reacts with indole to form a red or pink ring at the interface, indicating a positive result for indole production.

• E. Coli

• Positive

  • pink or red ring: positive

• Negative

  • no color change

  • K. pneumoniae

PHENYLALANIN DEAMINASE PRODUCTION

Determines if the bacteria produces phelalanine deaminase which oxidatively deaminates phenylaline into phenylpyruvic acid and ammonia

• primarily used to differentiate gram negative bacteria

• Positive

  • formation of a green color

  • P. vulgaris

• Negative

  • yellow/no color change

  • K. pneumonia

UREASE PRODUCTION

Used to determine is a bacterium can produce urease which hydrolyzes urea into ammonia and carbon dioxide

• Phenol Red is used as a pH indicator

• Ammonia can be used by some bacteria as a nitrogen source and can protect organism from acidic envrionment

• Positive

  • Pink color indicates a positive urease reaction, confirming the presence of urease-producing bacteria.

  • pH increased

• Negative

  • orange or yellow color

  • pH stayed the same or did not increase

KLIGLER’S IRON AGAR (TSI) SLANT

Used to differentiate bacteria based on sulfur reduction and glucose, lactose, and sucrose fermentation

• inocculated by a fistail streak and then a stab into the slant at the top to ensure even distribution of bacteria throughout the medium, allowing for optimal growth and metabolic activity.

• Positive

  • Glucose ONLY

    • slant turns yellow for 24 hour and then turns red again

  • Glucose, lactose, and sucrose

    • slant stays yellow

    • K. pneumoniae

  • Gas production

    • Slant moves upwards in tube

      • K. pneumoniae

    • Hydrogen sulfate: black precipitate at the bottom

      • P. vulgaris

• Negative

  • no color change

  • no movement of agar

  • Pseudomonas aeruginosa

MACCONKEY AGAR (selective/differential agar)

Can be used to differentiate Gram Positive from Gram Negative Bacteria and separate lactose fermenters from nonlactose fermenters

• Positive

  • gram negative bacteria

  • Pink/Red colonies: lactose fermenters

  • Colorless colonies: nonlactose fermenters

• Negative

  • no growth

  • gram positive bactera

MANNITOL SALT AGAR (selective/differential agar)

Can be used to differentiate between Gram Positive and Gram Negative bacteria

• Selective for Staphylococcus species

• inhibits growth of non salt tolerant bacteria and gram negative

• Positive

  • Gram positive

  • staphylococcus aureus

• Negative

  • gram negative

  • no growth

  • K. pneumoniae

EOSIN METHYLENE BLUE (EMB) AGAR (selective/differential agar)

Can be used to isolate gram negative bacteria and fermentation of lactose and sucrose

• Positive

  • Green Metallic Sheen: vigorus lactose and sucrose fermentation

    • E. coli

  • Pink/Mucoid colonies: slow lactose fermation

    • K. pneumoniae

  • Colorless colonies: no ferementation of lactose or sucrose

    • Salmonella

• Negative

  • no growth

  • gram positive bacteria

  • Streptococcus species

BLOOD AGAR

Can be used to differentiate bacteria based on their ability to produce hemolysins and engage in hemolysis (destruction of RBCs)

Positive

• Alpha hemolysis: partial breakdown of red blood cells, resulting in a greenish discoloration of the agar surrounding the colonies.

  • streptococcus pneumoniae

• Beta hemolysis: complete lysis of red blood cells, leading to a clear zone around the colonies.

  • streptococcus pyogenes

Negative

• Gamma hemolysis: no lysis of red blood cells, indicated by no change in the color of the agar.

  • K. pneumoniae