Studied by 4 people
Dilution Problem, Physiological tests, Staining, Protists, Fungi, and Helminths
Know how to do dilution
Individual dilution
Total dilution
How much plating: 1.0 ml (x1) or 0.1 ml (x 10^-1)
(30-300) x DF
MacConkey agar (MAC)
Purpose: to differentiate between lactose fermenting G- bacteria and lactose non-fermenting G- bacteria
Medium: crystal violet and bile salts
Type of Medium: selective and differential
+ results = pink colonies
- results = no color change
significant specific ingredients:
reagents/indicators:
Crystal violet and bile salts inhibit Gram (+) bacteria
and neutral red dye stains microbes fermenting lactose (and thereby decreasing the pH) a pink color.
specific directions (if any): streak agar in a straight line and incubate
significant results:
Columbia Nalidixic Acid agar (CNA)
Purpose: for the isolation of G+ cocci
Medium: contains the antibiotics colistin and nalidixic acid
Type of Medium: selective and differential
Growth (G) = clearly visible, good growth
Weak growth (WG) = very little growth
No growth (NG) = no visible growth
significant specific ingredients:
reagents/indicators: colistin and nalidixic acid inhibit G- bacteria
specific directions (if any): streak agar in a straight line and incubate
significant results:
Catalase
Purpose: to detect the production of the enzyme, catalase
Medium: TSA
Type of Medium: differential
+ results = bubbling due to the release of oxygen via catalase
- results = no bubbling
significant specific ingredients:
reagents/indicators: 3% Hydrogen Peroxide (H2O2)
specific directions (if any): apply 3% hydrogen peroxide to growth from a TSA plate
significant results:
Oxidase
Purpose: to detect the production of the enzyme cytochrome c oxidase
Medium: use growth from a TSA plate or slant
Type of Medium: differential
+ results = color change to purple within about 30 sec
- results = no color change or a change after more than 30 sec
significant specific ingredients:
reagents/indicators: oxidase dry slides; used after growth on TSA.
specific directions (if any): using a sterile wooden stick, pick a colony of bacteria from a TSA plate or slant and touch an area on one section of the dry slide
significant results:
Methyl Red test (MR)
Purpose: to determine mixed acid fermentation (lactic, acetic, formic, etc)
Part of the IMViC tests.
Medium: MRVP broth--buffered peptone glucose broth
Type of Medium: differential
+ results = red
- results = yellow
weak + = orange
significant specific ingredients:
reagents/indicators: methyl red (added AFTER incubation)
specific directions (if any): broth is inoculated and incubated. After incubation, add 5 drops of Methyl Red indicator, do not shake the tube, and read the results immediately
significant results:
Voges Proskauer test (VP)
Purpose: to detect the production of acetoin or butanediol from the fermentation of glucose in the broth
Part of the IMViC tests.
Medium: MRVP broth-- buffered glucose peptone broth
Type of Medium: differential
+ results = red layer at the top in 10 minutes (earliest detection), progressing downward
- results = no red color, disregard any copper or brownish-purple color
significant specific ingredients:
reagents/indicators: Barritt’s reagents A and B added AFTER incubation
specific directions (if any): inoculate broth and incubate. After incubation add 20 drops of Barritt’s Reagent A and 20 drops of Barritt’s Reagent B. Vortex at frequent intervals and allow the reaction to develop for up to 1 – 2 hours.
significant results:
Phenol Red Broths - Lactose, Dextrose (Glucose), Sucrose
Purpose: to distinguish carbohydrate fermenters from non-fermenters & to detect and distinguish the different, specific carbohydrates by the products formed
Medium: 0.5% to 1% carbohydrate (dextrose/lactose/sucrose) broth, peptone, with phenol red and an inverted Durham tube for detection of gas.
Type of Medium: general purpose differential test media
+ results = turns from red to yellow
- results = no color change
Gas production (+) = bubble trapped in an inverted Durham tube
No gas production (-) = no bubble trapped in an inverted Durham tube
significant specific ingredients:
reagents/indicators: phenol red as a pH indicator to indicate acid production
specific directions (if any): inoculate tubes and incubate
significant results:
AG = acid with gas production
A = acid, no gas
(-) = negative for acid and gas
Simmon's Citrate test
Purpose: to determine an organism’s ability to use citrate as the sole source of carbon
Part of the IMViC tests.
Medium: Simmons Citrate Agar- contains sodium citrate as the sole carbon source, mineral salts, and pH indicator Bromothymol blue
Type of Medium: selective and differential
+ results = color change from green to blue
- results = no color change
significant specific ingredients:
reagents/indicators: Bromothymol blue is a pH indicator
specific directions (if any): streak slant, cap loosely, and incubate.
significant results:
Starch hydrolysis
Purpose: to detect the production of the enzyme amylase
Medium: Starch Agar plates (1% starch)
Type of Medium: differential
+ results =Â clear zone around growth
- results = no zone
significant specific ingredients:
reagents/indicators: Gram’s Iodine
specific directions (if any): streak agar in a straight line and incubate. After incubation, add Gram’s iodine, dropwise, sparingly, just to cover growth and surrounding area on the medium.  Let the plate sit for a few minutes for the reaction to develop
significant results:
Casein hydrolysis (Skim Milk)
Purpose: to detect the production of the enzyme casease
Medium: Skim Milk Agar
Type of Medium: differential
+ results = clear zone around growth
- results = no clear zone
significant specific ingredients:
reagents/indicators:
specific directions (if any): streak agar in a straight line and incubate
significant results:
Urease production
Purpose: to detect the production of the enzyme urease
Medium: urea broth
Type of Medium: differential
+ results = red or bright pink color
- results = yellow color
significant specific ingredients:
reagents/indicators: Phenol Red
specific directions (if any): inoculate urea broth and incubate
significant results:
Gelatin Liquefaction
Purpose: to determine the production of gelatinase
Medium: Nutrient Gelatin Deep (12- 15% gelatin)
Type of Medium: differential
+ results = Liquefaction (after refrigeration)
- results = Gels when refrigerated, no liquefaction
significant specific ingredients:
reagents/indicators:
specific directions (if any): deep stab inoculation and incubate. After incubation, refrigerate for 1 hour before reading.
significant results:
Sulfide, Indole, Motility (SIM) Medium
Purpose: to differentiate G- rods
Medium: contains casein peptone, ferrous ammonium sulfate, sodium thiosulfate, and agar
Type of Medium: selective and differential
+ results:
Sulfide (+) = blackening of the media
Indole (+) = pink to red color observed on the surface after the addition of Kovac’s reagent
Motility (+) = growth observed beyond the stab line (cloudy, turbid)
- results:
Sulfide (-) = no blackening of the media observed
Indole (-) = yellow color observed on the surface after addition of Kovac’s reagent
Motility (-) = no growth observed beyond stab line (crisp solid inoculation line visible)
significant specific ingredients:
reagents/indicators:
ferrous ammonium sulfate and sodium thiosulfate are present to detect hydrogen sulfide (H2S) production
The agar in the medium creates a semi-solid environment for motility to be visualized
Kovac’s reagent (added AFTER incubation) will detect indole production
specific directions (if any): inoculate the media using your needle, stabbing down the center of the media in the tube
significant results:
IMViC tests
(Indole, Methyl Red, Voges-Proskauer, Citrate)
a battery of tests to help identify enterics and other GNRs
Negative staining
Negative stains are when we use Nigrosin (an acidic stain → it has a neg. charge) to stain bacteria and view them in their original shape and size due to the lack of heat fixation. The negative charge of the stain repels the negative charge of the bacteria’s cell, making it appear clear.
A negative stain is very good for observing cell morphology, arrangement, and size because the slide is NOT heat-fixed.
Simple staining
A simple stain is when we use a positively-charged dye to stain the negatively-charged bacteria and view them easily under the brightfield lens
Gram staining
We might use a differential stain in a mixed sample of organisms to differentiate cell types. Bacterial cells with gram-positive cell walls are stained purple with the dye crystal violet, while bacterial cells with gram-negative cell walls are counterstained pink with safranin dye.
Endospore staining
An endospore is a structure for the preservation of the DNA while conditions are poor
The heating process makes the spore coat more permeable to the stain
Endospores are formed when the bacterium is stressed.
Endospores can be seen in simple and Gram stains, as a clear or empty space in a vegetative cell.
Acid Fast staining
In the Acid-Fast stain, a very concentrated stain solution of kinyoun carbolfuchsin (hot pink) is used in order to penetrate and stain the cell walls
This stain is a valuable diagnostic tool for finding tuberculosis and nocardial diseases in patient specimens
Mycobacterium smegmatis is acid-fast positive
Smear prep from liquid or solid
(photo)
Good smear versus poor smear
A good smear is evenly distributed and not too thick or sparse.
If the smear is too thick the bacteria will not stain evenly. A thick/uneven smear cannot be properly decolorized and might wash off the slide despite fixation; we also won’t be able to see the bacteria clearly (morphology and arrangement) if they’re all piled on top of each other.
Heat fixation
We use heat to fix the bacteria onto the slide so it doesn’t wash away with the staining agents and DI water.
Immersion oil
A drop of immersion oil is used ONLY on the 100x objective lens to view microorganisms more clearly since the oil increases the resolving power
Immersion oil has the same refractive index as glass
How to observe differential stains
By always using brightfield on a 100X objective lens with oil immersion
What are the general characteristics of the protists?
eukaryotic and unicellular
Compare and contrast algae versus protozoa
Protozoa: most are harmless free-living in a moist habitat but some are animal parasites; malaria and toxoplasmosis
Algae: free-living; photosynthetic; causes Harmful Algal Bloom and Paralytic Shellfish Poisoning
What is the difference between a trophozoite and a cyst?
Cyst = the dormant stage; very resistant structures that allow protozoa to survive in adverse environments
Trophozoite = active feeding stage; absorbing nutrients from the host
Why would we use a hanging drop slide?
preserves cell shape and arrangement
the Vaseline-sealed depression also slows down the drying-out process, so the organisms can be observed for longer periods.
Plasmodium vivax
S.G. Chromalveolata, causes malaria, parasitic
Giardia
S.G. Excavata, flagella, diarrheal disease, parasitic
Entamoeba histolytica
S.G. Amoebozoa, pseudopods, diarrheal disease, parasitic
intestinal amebiasis, a very common protist infection worldwide, can survive hours in a pool and only needs 10 cysts to infect
Trichomonas vaginalis
S.G. Excavata, flagella, STI, parasitic
very common STI; doesn’t form cysts so needs to go from “ideal” wet environment to the next via sex
Cryptosporidium
S.G. Chromalveolata, diarrheal disease, parasitic
can survive in a chlorinated pool
What are the major characteristics of fungi?
chemoheterotrophic organisms that are usually nonmotile and grow by absorbing nutrients from their surroundings
Descriptions of molds versus yeasts
Yeasts are single-celled while molds are multi-celled (made up of hyphae)
Hyphae and mycelia
hyphae = long, threadlike filaments
mycelia = mass of hyphae
Types of sexual spores - what are the differences?
Ascomycota → ascospores
Basidiomycota → basidiospores
Zygomycota → zygospores
Types of asexual spores - what are the differences? (sporangiospores, conidia)
conidia = not in a sac, extend from the hyphae
sporangiospores = enclosed in a sac
Candida albicans
Sexual: Ascomycota
Asexual: budding?
Lives on your body (skin, GI and vaginal tract, & oral cavity)
dimorphic
septate
Candidal Intertrigo (superficial skin-fold infection) and Candida “Thrush” (overgrowth in the mouth and throat that appear as white, raised bumps)
Coccidioides immitis
Sexual: Ascomycota
Asexual: segmentation of hyphae produces arthrospores and can be inhaled?
Windy conditions spread its fungal spores easily; septate
dimorphic
saprophytic
septate
Valley Fever
Rhizopus
Sexual: Zygomycota
Asexual: sporangiospores
dimorphic
saprophytic
aseptate
bread mold
Aspergillus
Sexual: Ascomycota
Asexual: conidia
Very common airborne soil fungus
dimorphic
septate
“Farmer’s Lung”
Penicillium
Sexual: Ascomycota
Asexual: conidia
dimorphic
septate
General characteristics of the helminthes
They’re parasitic worms that are multicellular, eukaryotic, invertebrate animals, most are macroscopic but have microscopic life stages
Phylum Platyhelmintes
Class Trematoda
flukes, leaf-shaped, intermediate hosts are mollusks, definitive hosts are vertebrae, digestive system
Class Cestoda
tapeworms, long/ribbon-like bodies, scolex for attachment, two or more hosts, no digestive system
Phylum Nematoda
roundworms, cylindrical, one host, complete digestive system, diverse and numerous
What is a scolex and a proglottid?
The scolex is essentially the head, which contains organs which facilitate attachment to the host tissue
Proglottids are segments of the worm’s “neck” which contain eggs
Taenia solium
Class Cestoda, proglottid, tapeworm
Schistosoma
Class Trematoda, blood fluke, can burrow into the skin, urine (and fecal) contamination, snails = intermediate host, not hermaphroditic, live in water
Clonorchis sinensis
Class Trematoda, oriental river fluke, parasitic, obtained by eating undercooked fish
Enterobius vermicularis
Nematoda, pin worm, most common in U.S., mostly affects kids, very good at lasting in the environment; at night, the worm migrate to the anus and lay eggs
Ascaris lumbricoides
Nematoda, most common in the world, fecal/oral route, ingest egg → turns to larva → burrows out of intestine → pulmonary artery → lungs → clear throat → ingest again
Necator americanus
Nematoda, hookworm, live in humid/swampy areas, burrow into your skin (in between your toes)
Note 
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