Mystery Microbe

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Introduction: Being able to identify a particular bacterial species is important. It is very useful in knowing its risk of toxicity to humans or animals, its resistance or susceptibility to antibiotics, and determining how to control its growth or kill it altogether. The purpose of these procedures is to discovery the identity of an unknown microbe by observing its reactions to a barrage of chemical and physical tests. Different microorganisms react in different ways, due to their function, digestibility, morphology, chemical make-up and other details. By observing the responses to these tests performed in a particular sequence, some can be eliminated as possibilities and others require further investigation.

Materials and Methods: A stock culture, labelled 25, was stored at room temperature (in the lab desk). A transfer loop was sterilized and a sample of the stock was transmitted to a tube of sterile BHI broth (for a working culture) and also to an agar plate and incubated for 24 hours. From the incubated plate, distinct and well isolated waxy, yellowish colonies appeared. Another sample was applied to a glass slide, heat fixed and a Gram stain test was performed. From the list of several possible choices for the unknowns, about half are eliminated. Table 1 indicates the tests that were performed on the unknown sample, all materials required for each test, as well as factors determining a positive or negative result. Test PerformedMedia/Reagents UsedPositive Test ObservationsNegative Test Observations Gram StainCrystal violet

Gram’s iodine
95% Alcohol
SafraninBlue or violet cellsPink cells
Catalase Glass slide
3% H2O2
Agar plates with colonies
ToothpickBubbles producedNo bubbles produced
OF Test2 tubes of OF medium (glucose agar with bromthymol blue) 1 tube with sterile oil
1mL pipette
Pipette and pumpColour change from blue to yellow in tube covered with oil (F)Blue/no colour change in tube covered with oil (O) or both (–) (does not use glucose) Sulfide-Indole-Motility1 tube of SIM agar

Erlich’s reagent
Inoculating needleS-Black precipitateS-No black precipitate
I -Red colour after addition of Erlich’s agentI-No red colour after addition of Erlich’s agent
M- Cloudiness in tube, movement from stab lineM-No swarming from stab line, no cloudiness

MR/VP1 tube of MR-VP medium (glucose broth with buffered peptone and K2PO4) Empty test tube
1mL pipette
Pipette pump
Alpha napthol
KOH solution
Methyl RedReddish colour/red ring after standing for 30 minutes. Reddish colour results in acid end productsNo appearance of red colour. No colour change, yellow neutral end products Nitrate1 tube of trypticase-nitrate broth

Sulfanilic acid (part A)
Dimethyl-alpha-naphthylamine (part B)
ZincRed colour after the addition of solution A and B
Cloudy grey colour after addition of zinc dustNo colour change after addition of solution A and B AND
Red colour after addition of zinc
Urease1 tube of urea slants (yeast extract, urea buffer and Phenol-red)Pink or purple colourYellowish/no colour change Degradation of Glucose and other Simple SugarsPhenol-red glucose broth Durham tube Phenol-red sucrose broth Durham tube

Phenol-red lactose broth Durham tube
Wire loopColor change from red to yellow (acid production) and bubble trapped in Durham tube (gas production) Red color remains, no bubble in Durham tube
Starch hydrolysis1 starch/agar plate
Gram’s iodineClear zone among the blue plate, after application of iodine Blue colour across the entire plate Casein hydrolysis1 agar plate with caseinClear zone around the streakNo clear zone around the streak Gelatin hydrolysisNutrient gelatin deeps

Straight wire inoculating needle
FridgeGelatin becomes liquid, despite refrigerationGelatin is gelled after refrigeration Table 1: Tests performed during investigation, media/reagents/equipment used,...
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