Biol241L
Clara Tsui
Experiment 1 and 2-milk
Milk contains: gram-positive, nonmotile, microaerophilic/anaerobic rods/cocci
Ie. Lactobacillus, Micrococcus, Streptococcus
- direct plate count
- direct microscope factor calculation  area of field (πr²)  convert from mm² to
cm²
o # of fields in 1 cm²/area = # of fields
o Microscope factor = # of fields / vol. of milk over 1 cm²
o (in our experiment it was 0.01ml/cm²)
Fresh pasteurized milk had the lowest count; past-date is next, then raw milk has most
Standard plate count  colonies formed = # of viable microorganisms
(only the MOs capable of growing on those nutrients/conditions)
*pour plate technique was used
Exp 3- cheese
MEA- selective (mold/fungi can grow  detect cheese mold)
EMB – detect coliforms that cause gas defects in cheese
- differential  E. Coli – green metallic sheen around dark purple colonies
 E. aerogenes – pink mucoid colonies
- selective  methylene blue and eosin inhibits growth of gram-positive organisms (ie.
Streptococcus, staphylococcus)
APT- non selective
Streptococcus and Lactobacillus are lactic-acid producing bacteria (initiate cheesemaking process)
Defect: Clostridium/E. coli/Enterobacter aerogenes form gas, or Geotrichum,
Penicillium form dairy mold
Exp 4 – Food illness by Staphylococci/Salmonellae
Salmonella not usually found in food products, so enrichment step is necessary;
staphylococci are abundant
- usually in food poisoning outbreaks, only one organism is the agent
Staph: positive coagulase test
Salm: selenite-cystine=enrichment (in real life, tetrathionate brilliant green is too); plate
on MacConkey agar ensures isolated colonies (brilliant green sulfa used IRL)
- g+ve org inhibited; lactose fermenting =red/dark pink, zone of precipitated bile
- non-lactose fermenting  pick transparent-pink colony to TSI slant
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Clara Tsui
Obs: red = negative for lactose/sucrose utilization; black = H2S production, dextrose
utilization; gas formation positive
Lactose use (+ve)  yellow
Exp5 – MPN Test (Coliforms in water)
Water always has large # of MOs, but it is potable as long as human pathogens are
absent
- test for coliforms (MOs indigenous to human intestinal tract that are rarely found
in env’ts other than feces)
(1) presumptive
- multiple lauryl tryptose broth tubes  gas production? YES = unsafe
(2) confirmed
- brilliant green lactose bile (selective for coliforms)
- inoculate with YES tubes from (1)
- gas production = positive
- alternate: streak onto EMB or Endo agar, look for greenish metallic (E.Coli) or
pink w/dark centre (E. aerogenes) colonies
(3) completed
- inoculate the coliform colony into lauryl tryptose broth and make g-stain slide
- g-negative, nonsporeforming bacilli that produce acid+gas from lactose =
coliforms
Exp6 – Membrane filter technique
ENDO medium: basic fuchsin + aldehyde = shiny green complex (= coliform, since
they break down lactose into simpler substances)
Total coliforms  incubate at 35°C
M-FC: fecal coliforms are blue; incubated at 44.5°C
Method is sensitive and large volumes of water can be tested, shorter test time, but filter
can be plugged easily by cyanobacteria
Exp7
B. subtilis and S. aureus are not coliforms b/c they don’t grow on EMB or Endo
Exp8 – IMViC Test
I=Indole production
Biol241L
Clara Tsui
- tryptophan  NH3 + pyruvic acid + indole ring (by tryptophanase)
- indole ring + Kovac’s = red
M=Methyl red test
- glucose+peptone water to see if MO ferments glucose  pyr. Acid
- red = +ve (pH<4.5 pyruvic acid produced)
- yellow = -ve (pH>4.5 peptone use after glucose exhausted raises pH)
Vi=Voges-Proskauer reaction
- glucose  pyr acid  acetonin (acetylmethylcarbinol)
- Acetonin + alpha-naphthol + KOH = red
- Negative = brown
C=ability to use citrate as sole carbon source
- Positive = can use sodium citrate, shift pH to basic side
- Indicator bromothymol blue = blue, from neutral green/yellow (acid)
Exp9-Chlorine demand
- reagent: ortho-tolidine
- chlorine is a strong oxidizing agent; marked affinity for organic cpds
- water supplies w/organic matter have “chlorine demand”
- only available chlorine (not Cl used by demand) is bactericidal
- peptone=affinity for Cl so some growth is observed, more than just bacteria+Cl
but less than no chlorine added
Exp10-Oxidation of Carbon source
- NaOH titrated with HCl to determine remaining amount
- NaOH + ½ CO2  ½ Na2CO3 + ½ H2O
- n=c*v
Exp11-Nitrification
Nitrosomonas – NH4+  NO2Nitrobacter – NO2-  NO3Ammonia detection: Nessler’s reagent (positive = yellow colour/brown precipitate)
Trommsdorf’s reagent + sulfuric acid: nitrite test (brown-block = positive)
Diphenylamine+sulfuric acid : nitrate test (black = poisitve)  only valid if
Trommsdorf test is negative (detects nitrite and nitrate)
Exp12-Denitrification
-anaerobic
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Clara Tsui
NO3-  ½ N2 + 3 H2O
Pseudomonas; look for production of gas in tryptone-yeast-nitrate-broth
Exp13-Ammonification
nitrogen is contained in coenzymes/proteins/nucleic acids
proteases=extracellular hydrolytic enzymes
Nessler’s, bromothymol blue, urea use
Exp14-Nonsymbiotic Nitrogen Fixation
Only a few bacteria + cyanobacteria can fix nitrogen (atmospheric  available to
organisms)
Azotobacter = aerobic soil org; gram-negative, produces capsular slime; forms thickwalled cysts; +/- water-insoluble melanin pigment and/or water-soluble fluorescent
pigment (appears green under UV)
Clostridium pasteurianum = anaerobic
Rhodospirillum rubrum = photosynthetic (important in nitrogen enrichment of soil)
Ammonia + alpha-ketoglutaric acid = glutamic acid (very versatile aa)
Azomonas – large ovoid cells in pairs/clumps (like Azotobacter), some can N2 fix;
however does not have large cysts, appears white under UV light
Morphology
Chroococcum Large ovoid rod, pairs
Beijerinckii
Large ovoid rod,
single/pair/chains
- coccoid in older cultures
- has large capsules
Vindelandii
Large ovoid rod, pairs
Paspali
Large ovoid/pleomorphic rod
- variations from coccoid to
elongated filaments occur
Pigment
Fluorescence
Brown/black
None
Yellow/cinnamon None
in older cultures
None
None
Yes, green
Yes, green
Exp15-symbiotic N2-fixing bacteria
Nodules are formed of thin-walled parenchyma cells packed with Rhizobia
- rods
- change to x and ys (bacteroids)
- gram negative (stain pink)
Exp16-Sulfur oxidation
Biol241L
Clara Tsui
2S + 3 O2 + 2 H2O  2 H2SO4
Thiobacillus, Thiobacterium, Thiospira
Found in sewage, sulfur springs, coal-mine drainage, polluted stagnant aquatic env’ts
pH decrease = sulfur oxidation; growth can occur at 1, grows readily at 3!
Exp17-sulfate reduction
SO42-  SO32-  S2- (H2S)
Desulfovibrio
ATCC medium #2 contains ferrous ammonium sulfate  blackening = reduction
Fe(NH4)2(SO4)2  S2- + Fe  FeS
Exp18-Bacteriophage assay
Viruses: nucleic acid (RNA OR DNA) + protein outer covering (coat/capsid)
Polyhedral head (65x95 nm) and contractible tail (17x115 nm)
Bacteriophage host specificity: phage for one strain of bacteria within a species may not
attack another strain
Lytic cycle-replication of more phage particles + destruction of cell w/release of virions
Virulent phage-phage that attaches to cell envelope
Vegetative state – phage nucleic acid is inside cell
Empty coat – ghost
Burst size = # of particles liberated per host cell
Latent period = period from vegetative  liberation of new virions
Turbidity decreases!
Plaque = clear area produced by destruction of cells
* each plaque initiated by single virion; one virion is adsorbed onto a single host cell
Exp19 – Microbial symbiosis in lichen
Association of alga/cyanobacterium + fungus
Can grow at low temps or extreme env’ts (on rocks/bark/other usually unsuitable
substrates)
Thallus = tightly woven fungal mycelia
Below: layer of algal cells
Bottom: attaches to substrate directly or by short twisted strands of hyphae
(rhizomes/rhizoids)
Fungal partner = mycobiont (usually associated form, not free living)
 provides mechanical support/protection for alga, or inorganic nutrients req’d by alga
Biol241L
Clara Tsui
 stores water = microenv’t high in water for alga
Algal component = phycobiont (if it is cyanobacteria, N2 fixation may be possible!)
 can be free-living
 photosynthetic, metabolize CO2 + water to make materials available to mycobiont
(carbs/vitamins)
Green algae produces sugar alcohols: ribitol, erythritol, sorbitol
Cyanobacteria  glucose
Fungus  other sugar alcohols (arabitol, mannitol), utilized as energy source
Components: (should not go beyond 10x objective)
a) upper cortext (surface layer of fungus)
b) algal layer
c) medulla (filamentous portion of fungus)
d) substrate layer of fungus
Exp20-Multiple test/rapid method systems
API 20
BBL Crystal Enteric/nonfermentor ID system
Value # * 1/0 (+/-)  add 3 together to get # between 1-7
7 digits form the profile #
Incubate to ensure fermentation, not oxidation
Sterile mineral oil = seal out air
Motility: hanging-drop
Oxidase (transfers e-‘s to oxygen): reagent = 1% dimethyl-p-phenylenediamine HCl 
purple = +ve
-ve = clear/light purple
Continue w/API test if oxidase + and GLU – (blue/green)
GLU tube is positive: VP test, indole
Oxidase +ve  nitrate reduction test (add sulfanilic acid +
dimethylalphanaphthylamine; red=positive; reconfirm neg by adding zinc dustred; if
no colour change upon adding zinc dust, + for nitrate reduction)
Biol241L
Clara Tsui