Path Micro Lab Final

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Methyl Red

- Test to determine if an organism uses the mixed acid fermentation pathway for glucose
- Uses a pH indicator
- Strong acids will be produced and the pH of the medium will be below 4.4 and the reagent will turn red when added.

Methyl Red Protocol

- Inoculate broth with organism
- Incubate for 48 hrs.
- Add 3-5 drops of MR reagent.
- Read results
- Red = (+)
- No color change = (-)

Voges-ProskauerTest

- Test to determine if an organism ferments glucose via the 2,3-butanediol pathway.
- Acid by-products from this pathway are less stable and are quickly converted to more neutral products such as acetoin and 2,3-butanediol.
- Detects acetoin (also called acetylmethylcarbinol).
- The reagents oxidize the acetoin to diacetyl which can complex with guanidine nuclei from the peptone (in the medium) to form a brick red color.
- If after the addition of reagents a brick red is seen, then the organism uses the 2,3-butanediol pathway to ferment glucose.

Voges-ProskauerTest Protocol

- Inoculate broth with organism
- Incubate for 48 hrs.
- Add 15 drops of VP I (alpha-naphthol) and mix well
- Then add 5 drops of VP II (KOH) and mix well again.
- Put the tubes back in the rack and do not disturb until you read results.
- Read results (1/2 hr. - 1hr).

Citrate Test

- Test used to determine if the organism can utilize sodium citrate as its sole carbon source.
- The medium contains ammonium phosphate as its sole nitrogen source.
- Bromthymol blue is the indicator and is green at pH 6.7 and blue at pH 7.6.
- Organisms that can utilize citrate will also convert the ammonium phosphate to ammonia and ammonium hydroxide, raising the pH and turning the medium blue.

Citrate Test Protocol

- Lightly inoculate the surface of the slant with a single streak up the slant.
- Incubate for 24 hrs.
- Blue color = positive.
- Growth with no color change is also considered positive (continue incubation if you think this is the case).

Malonate Test

- Test used to determine if the organism can utilize sodium malonate as a carbon source.
- Bromthymol blue is the indicator
- If the organism is able to utilize malonate the medium will become alkalinized and it will turn blue (positive).
- A negative result can be no color change (green) or possibly if the small amount of glucose that is in the medium was fermented it could be slightly yellow.

Malonate Test Protocol

- Inoculate organism into malonate broth.
- Incubate 24 hrs.
- Blue = positive
- Green or slight yellow = negative

Decarboxylation of Amino Acids

- Test that uses Moller's Decarboxylase Base Medium, which contains glucose, the specific amino acid and bromcresol purple as the pH indicator.
- If organism is able to decarboxylate the amino acid (remove a carboxyl group, COOH), it will create amine byproducts that will raise the pH of the medium making it purple.
- Glucose is added to the medium because decarboxylase enzymes are activated at an acidic pH and under anaerobic conditions.
- After inoculation, a layer of mineral oil is added on top of the medium to create an anaerobic environment.
- If fermentation of the glucose takes place (yellow), then an acidic environment is created which can activate the decarboxylase enzyme which will create an alkaline environment.
- Purple starting color → yellow (fermentation, acidic environment to activate decarboxylase) → back to purple = decarboxylation took place (positive result).

Decarboxylation of Amino Acids Protocol

- Inoculate organism into the lysine, arginine and ornithine media as well as the control medium (control contains no amino acid).
- Aseptically pour a layer of mineral oil on top of each of the media.
- Incubate 24 hrs.
- Purple = positive result for decarboxylation of amino acid.
- Yellow = negative result for decarboxylation of amino acid.

Phenylalanine Deaminase Test

- Test used to determine if the organism can produce phenylalanine deaminase (PD).
- If organism can produce PD, then the phenylalanine in the medium will be deaminated (amine group removed) and ammonia and phenylpyruvic acid are produced.
- Ferric chloride is the reagent added to the slant medium after incubation and can react with the normally colorless phenylpyruvic acid to produce a green color.
- Only Proteus, Providencia and Morganella will have a positive PD test of all the Enterobacteriaceae

Phenylalanine Deaminase Test Protocol

- Inoculate the phenylalanine slant (streak up the slant)
- Incubate for 24 hrs
- Add a few drops of 10% ferric chloride, let it run down the slant.
- Development of green color = positive
- No green color change/yellow = negative result.

Motility Test

- Test allows for identification of motility.
- Medium is a semisolid to allow for movement of the organism through the medium.
- Medium contains a tetrozolium salt to aid in the identification of motility.
- The tetrazolium salt turns red in the presence of an metabolizing organism.
- If there is motility there will be a red color change away from the inoculation line
- If no motility then the red color will only be along the inoculation line.

Motility Test Protocol

- Inoculate each of the organisms, using a straight up and down motion (stab)
- Incubate 24 hrs.
- Migration away from inoculation line = positive.
- No migration away from inoculation line = negative.

Fermentation of Inositol and Adonitol Test

- Tests for the fermentation of inositol and adonitol to help speciate members of Providencia.
- Bromcresol purple is the pH indicator and is purple above pH 6.8 and yellow below pH 6.8.
- If organism is able to ferment the sugar, then the medium will turn yellow.

Inositol and Adonitol Test Protocol

- Inoculate both inositol and adonitol broths
- Incubate 24 hrs
- Yellow = positive
- Purple = negative

Urease Test

- Test used to determine whether the organism can make the enzyme urease.
- Urea broth contains urea and phenol red as the indicator.
- If organism makes urease, it will be able to hydrolyze urea to ammonia and water, thereby raising the pH to above 8.4 and turning the phenol red to a cerise (hot pink) color

Urease Test Protocol

- Inoculate organism into urea broth.
- Incubate 24 hrs
- Cerise = positive
- No color change = negative

Serratia marcescens

- Most common and clinically relevant of the species in the genus Serrtia.
- Can be found as normal flora of humans and is found in the intestinal tract.
- Can cause opportunistic infections
- Is a problem in the hospitals (nosocomial infections).
- Has been found to cause infections associated with surgery, blood transfusions and the urinary tract.

Citrobacter

- Contains several species with C. freundii and C. diversus being the most common.
- Are normal flora in the intestine of humans and like S. marcescens can cause extraintestinal infections

Orthonitrophenyl-β-D-Galactopyranoside Test (β-Galactosidase Test)

- Test used to identify the presence of the enzyme β-galactosidase. This enzyme hydrolyzes lactose into glucose and galactose once lactose is inside the cell
- Test is used to determine whether the organism produces the enzyme even if it appears the organism is not a lactose fermenter.
- Some organisms are deficient in another important enzyme called permease. This enzyme allows the bacteria to get lactose into the cell so that β-galactosidase can hydrolyze it.
- ONPG will detect β-galactosidase independently of permease, so if the organism has β-galactosidase it will cleave the lactose analog (ONPG) and create a yellow color.
- This will separate the non-lactose fermenters from the slow lactose fermenters.

Orthonitrophenyl-β-D-Galactopyranoside Test Protocol

- Put an ONPG-impregnated filter disc in 0.5ml of sterile saline.
- Inoculate heavily.
- Incubate 1-6 hours.
- Yellow color change (+) = β-galactosidase produced = lactose fermenter
- No color change (-) = no β-galactosidase produced = non-lactose fermenter

Deoxyribonuclease (DNase test)

- Test used to identy organisms that produce the enzyme DNase.
- The enzyme degrades DNA and in this test a green dye has been complexed with the DNA in the medium so if the DNA is degraded the dye is released and a clearing around the colony is seen.
- Room temperature

Deoxyribonuclease (DNase test) Protocol

- Do a single streak line down the plate.
- Incubate 24 hours at room temperature
- Clearing around the colony = (+)
- Red line = (+) for Serratia
- Growth but no change = (-)

Arabinose Fermentation Test

- This is an important test in identifying S. marcescens from the other Serrtia as well as Enterobacteriaceae.
- Almost all members of Enterobacteriaceae can ferment L-arabinose, with an important exception being S. marcescens.
- Bromcresol purple is the pH indicator and is purple above pH 6.8 and yellow below pH 6.8.
- If the organism is able to ferment the sugar, then the medium will turn yellow.

Arabinose Fermentation Test Protocol

- Inoculate the L-arabinose broth with organism
- Incubate 24 hrs.
- Yellow = positive.
- Purple = negative (S. marcescens)

Salmonella

- Found in humans and almost all animals around the world.
- Is always considered a pathogen if found in humans
- Some species can causes food poisoning from either improperly cooked poultry or improperly handled contaminated eggs.
- An organism is placed into this genus if it displays the following biochemical pattern: no fermentation of lactose or sucrose, produces H₂S, decarboxylates lysine, is motile and urease negative.
- Confusion can arise because Proteus will look very similar in their biochemical patterns on MAC and TSI so a urease test is used to distinguish between the two (Proteus is positive while Salmonella is negative)

Shigella

- Is always considered pathogens if found in humans.
- Human pathogens that cause bacillary dysentery (diarrhea that contains blood, leukocytes and mucus).
- The bacteria are transmitted person to person or via contaminated food or water.
- Displays the following biochemical patterns: no fermentation of lactose or sucrose, fermentation of glucose without gas formation, H₂S negative, lysine negative and non-motile.
- The key reactions are that they are non-motile and non-lactose fermenters.
- To separate S. sonnei from the other Shigella species the ONPG and ornithine tests are used.
- S. sonnei will be positive for both.

Four Tests of Initial Grouping of Nonfermentative, Gram-Negative Bacilli

1) glucose metabolism
2) oxidase reaction
3) ability to grow on MAC agar
4) motility

Hugh-Leifson oxidative-fermentative (OF) medium

- Allows differentiation of organisms according to how they metabolize glucose (oxidative, fermentative, or nonsaccharolytic (inactive))
- Oxidative bacteria don't produce a lot of acid and this acid reaction will easily be overcome by the breakdown of high peptone (amine byproducts) in TSI
- OF medium is low in peptone (0.2%) and high in carbohydrates (1%) so that the creation of acids by oxidation of the carbohydrates can be seen**
- The typical carbohydrates tested are glucose, maltose and xylose.
- Two tubes are needed, one "open tube" and one "closed tube" (oil overlay ~ 1 cm).
- The open tube will detect oxidation while the closed tube will detect fermentation.
- Bromthymol blue is the indicator
- A positive result is a yellow color change (acidic conditions) from the original green.
- This color change will generally take place at the surface first (oxidative).

Oxidative-Fermentative Medium Protocol

1) Stab two tubes of OF glucose, OF maltose or OF xylose media
2) Overlay one of the OF glucose tubes with ~ 1 cm of mineral oil (closed tube).
3) Incubate at 35°C and view each day for 3 days to see rxn

Fermentative Organism

OF open = (+) yellow
OF closed = (+) yellow

Oxidative Organism

OF open = (+) yellow
OF closed = (-) green

Nonsaccharolytic Organism

OF open = (-) green
OF closed = (-) green

Pseudomonas spp

- Most are oxidase (+) , grow on MAC, oxidative and motile by polar flagella.

Pseudomonas aeruginosa

- The most frequently isolated nonfermentive bacilli
- Produces a distinctive blue-green, diffusible pigment called pyocyanin.
- Pyocyanin is distinctive because P. aeruginosa is the only species that produces it.
- It can also produce another pigment called fluorescein, which will fluoresce under UV light.

Pseudomonas fluorescens

- Produces a pigment called fluorescein, which will fluoresce under UV light
- Can be confused because P. aeruginosa because it also produces fluorescein.
- The way these two organisms are differentiated is by growth at 42 degrees Celsius**
- P. aeruginosa will grow at this temperature while P. fluorescens will not**

Xanthomonas maltophilia

- Second most commonly isolated nonfermentive bacilli
- Grows well on MAC and is motile
- Differs in that it is oxidase (-), oxidizes OF glucose very slowly and strongly oxidizes OF maltose (key reaction)**
- Smells like ammonia

Alcaligenes faecalis

- Grows on MAC, are oxidase (+) and are motile
- Motility is by peritrichous flagella**
- A flagella stain is needed to differentiate from Pseudomonas
- Has a fruity odor

Achromobacter xylosoxidans

- Grows on MAC, are oxidase (+) and are motile
- Motility is by peritrichous flagella**
- Oxidizes OF xylose after only overnight incubation but only weakly oxidizes OF glucose** (sometimes more than 48 hours for positive result)

Trypticase Soy Agar (TSA)

- Is a general purpose medium.
- This is inoculated and incubated (loosely capped) at 42°C for 3 days and viewed daily to observe reaction.
- Useful for differentiating Pseudomonas
- P. aeruginosa will grow and P. fluorescens will not to grow.

Trypticase Soy Broth (TSB)

- This medium is used for a wet mount preparation for motility between Alcaligenes spp
- 1) Inoculate organism intoTSB.
- 2) After incubation organism has grown up (incubation):
- Place a loop of organism in the center of a microscope slide.
- Place a coverslip over the wet mount.
- Focus on the edge of the coverslip with the microscope on low power and the condenser diaphragm almost all the way down and have the image slightly out of focus (increase in contrast at the expense of resolution).
- This should allow the best visualization of motility.

P Slant Agar

- Contains magnesium to enhance pyocyanin production.
- Pyocyanin is a diffusible, blue or blue-green, water-soluble, chloroform-extractable, nonfluorescent pigment.
- The only species know to produce pyocyanin is P. aeruginosa**
- Incubated at 35°C and view each day for 2 days for reaction.

F Slant Agar

- Is enhanced with casein and meat peptone for the production of fluorescein.
- Fluorescein is an organic luminescent pigment the emits a green-yellow fluorescence when placed under a UV lamp.
- Both P. fluorescens and P. aeroginosa produce fluorescein.
- Incubated at 35°C for 48-72 hours.

Fungi: Cellular Level

- Euks have nucleus and organelles
- Large cells: 10 micrometers and larger
- Branching filamentous growth
- Similar to algae, but has no chloroplasts (no photosynthesis)
- Simple cell wall or chitin polymer (no peptidoglycan)
- Most have aerobic respiratory metabolism
- Have mating types: (+) or (-). For example Mucor (+) or (-)

Fungi: Cultural level

- Tolerates a wide range of pH and osmotic pressure (meaning they can grow in acidic or dry conditions that would inhibit most bacterial spp)
- But has less tolerance for wide ranges in temperature and redox conditions
- Use selective media that has a pH of 4.8 - 5.0

Superficial mycosis

- The infection is topical (skin, hair, nails)
- Ringworm type infections of Tinea: athletes foot, jock itch, inflammation of the torso, scalp, etc.
- Candida albicans: vaginitis or thrush

Deep-seated (Systemic) mycosis

- Infection of the respiratory system
- Serious
- Coccidiomycosis
- Aspergillosis

Traits for the Laboratory Study of Fungi

1) Hypha structure: Septate (bamboo looking) or Non-septate
2) Loose filamentous growth that spreads: Molds
3) Colonies of single-cell fungi: Yeasts
4) Large macrosize forms: Mushrooms, Toadstools
5) Asexual spores: Sporangiospores (in a sporangium sac) or Conidiospores (not enclosed)
6) Sexual spores: Zygospores (not enclosed), Ascospores (in an ascus sac) and Basidiospores (upon a basidium but not enclosed)

Zygomycetes

- Mating types produce sexual zygospores
- Vegetative hyphae filaments: Non-septate, branched
- Asexual spores: Sporangiospores in sporangium (on apical end of hyphae)
- Examples: Mucor spp and Rhizopus spp

Ascomycetes

- Produces ascospores in a sac called an ascus after mating

Ascomycetes: Yeast

- Single cell
- Reproduce asexually by binary fission and budding
- Reproduce sexually by mating (cell would now be an ascus and the ascospores are seen in the cell)
- Often produce pseudohyphae when budding-on-budding produces a filament-like extension on the cell
- Examples: S. cervisiae

Ascomycetes: Molds

- Multi-cell filamentous
- Sexual mating types
- Vegetative hyphae are septate
- Asexual conidiospores in chains
- Examples: Penicillium spp and Aspergillus spp

Basidiomycetes

- Have septate hyphae that are compacted into large forms
- Forms and produces basidiospores

Deuteromycetes

- Are nearly indistinguishable from the septated, conidiospore-forming Ascomycetes
- This spp has an inability to demonstrate the capability of sexual reproduction and ascospore formation**

One-Point Inoculate Potato Dextrose Agar (PDA)

- 1-point inoculation in 3 locations of the media
- Invert plate
- Incubate at 30°C for 3 days minimum
- Useful for observation of the aerial mycelium and substrate mycelium

Viewing the Cellular Features of Mold Hypha and Asexual Spores Protocol

1) Add a moistened paper liner to the bottom of plate (keeps the small agar cube from drying out)
2) Place a V-shaped glass rod on the moist paper
3) Flame-sterilize a microscope slide and place atop the V-rod
4) Cut a 1.0cm² agar block from a PDA plate and place on the sterile slide
5) Inoculate the agar block with a mold suspension
6) Flame-sterilize a cover slip and place on top of the inoculated agar block
- NO inversion of plates for incubation!

Mating Experiment

- Inoculate one PDA plate with each mating type using a 1-point inoculation on opposite sides of the agar surface
- Incubate 30°C
- Examples: Mucor hiemalis (+) and Mucor hiemalis (-)

Yeast study

- Uses a 3-zone isolation streak method on a PDA plate
- Invert plates
- Incubate 30°C

Obligate anaerobe

Cannot grow in the presence of oxygen (gradient of this, some organisms are very sensitive while others are more aerotolerant)

Facultative anaerobe

Can grow well with or without oxygen present

Strict aerobes

Cannot grow in the presence of anaerobic conditions

When working with anaerobes, what is the first thing to check for?

- To see whether you are working with an obligate anaerobe or a facultative anaerobic organism
- To do this, set up experiements for both conditions

Identification of an aerobe consists of:

- Determining whether the organism is an obligate anaerobe or facultative anaerobe
- Determining the Gram stain morphology
- Biochemical tests to determine the spp of the organism

Clostridium

- The only genus of spore-forming bacilli
- If an organism is anaerobic, Gram (+) bacilli and forms spores it is Clostridium
- The spores' location indicates what spp it is:
- Central (spore is in the middle)
- Subterminal (not quite at the end)
- Terminal (at the end)

For Complete Identification of Clostridium:

1) Location of spores
2) Lecithinase
3) Lipase
4) Gelatin hydrolysis
5) Reaction in litmus milk
6) Indole
7) Carbohydrate fermentation using special anaerobic media
8) Determination of metabolic by-product using gas liquid chromatograph (GLC)

Clostridium sporogenes

- Subterminal spores
- Lipase (+)

Propionibacterium acnes

- Gram (+) anaerobic bacilli, non-spore forming
- Colonies will be tan or white/opaque
- Is the only Gram (+) non-spore forming anaerobic bacilli that is catalase (+) and indole (+)

Anaerobic Environment Conditions

- Normal air is 79% N₂, less than 1% CO₂ and 21% O₂
- But for an anaerobic incubation environment there should be 80-85% N₂, 10% H₂, 5-10% CO₂, and less than 1% O₂

Creating an Anaerobic Environment

- There are two methods for creating an anaerobic environment
- In both methods, a catalyst is used to rid the environment of residual oxygen (in our lab we used palladium)
1) Anaerobic chamber: where the researcher/technologist can put their hands into gloves and work within the chambe without introducing outside gases
2) Gas generator: in wich a gas generating system is used within a closed jar or bag

Gas-Pak System

- Creates an anaerobic atmosphere using anaerobic jars
- The inoculated media along with an indicator strip and the catalyst (palladium) is placed in the jars
- Distilled water is added to activate the catalyst and the jar is sealed
- If the anaerobic environment is created, the indicator strip will turn white within 2 hours and moisture (water) will accumulate in the jar
- The palladium catalyst reacts with Hydrogen and Oxygen to form water: 2H₂+ O₂→ H₂O

Gas-Pak System Protocol

1) Cut the top corner of the generator envelope
2) Add 10 ml of distilled water
3) Place the generator envelop and indicator strip in the jar
4) Seal the jar with the lid
5) CO₂and H₂should evolve, removing any excess O₂

Enriched Thioglycolate Broth

- An enriched medium that supports most anaerobic bacteria
- Thioglycate base is a reducing agent that helps rid the media of oxygen (except at the top of the broth = pink)
- The medium is autoclaved just before used to drive off any oxygen
- Growth will appear as turbidity

Egg Yolk Agar

- A medium that contains an egg emulsion in a blood agar base
- Is used for the lecithinase and lipase test (both of these enzymes break down lipids)
- The egg yolks provide the lecithinase and lipase
- The medium also has vitamin K and hemin to enhance the growth of the anaerobes
- A positive test for lecithinase will look like an opaque (whitish) zone under and around the growth of the colony
- A positive test for lipase will look like an oily sheen or pearly layer on the surface of the agar

Tryptone Broth

- Used to detect indole production
- Indicates that the organism produces tryptophanase

Tryptone Broth Protocol

1) Inoculate organism into tryptone broth
2) Incubate in an anaerobic jar
3) Add Kovac's to one week old incubated broth, record results

Litmus Milk

- Litmus milk contains lactose (sugar), casein (protein) and a pH indicator (litmus)
- If lactose is fermented, the pH will be acidic and the indicator will turn pink
- If no fermentation takes place then the indicator is purple or blue
- Litmus can act as an electron acceptor and become reduced in which case it loses its color so the medium will appear white
- Casein can be precipitated by acids or converted enzymatically into a clot
- The casein can be broken down proteolytically (proteolysis) causing a clearing of the medium (clearing)

Litmus Milk Results

1) Pink - Acid
2) Purple or Blue - No acid formed
3) Clot - Coagulation of milk from fermentation of lactose
4) White - Reduction of litmus
5) Proteolysis - Clearing of medium

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