Biology 120: Microbiology (Laboratory)Identification of Unknown Bacteria

Casela, Joenilyn Patricia; Galan, Charize Mae, Gatchalian, Ysrael Orlando; Glory, Brendalyn; Villena, Juan Paolo DavidWAD1Dra. Josephine Bautista-Guerrero

I. Cultural Characteristics

Identify the following: Color of bacterial growth Approximate size of unknown Shape of colony growth Elevation of colony Margin of colony Surface texture

II. Morphological Characteristics (Cellular level)

Three basic shapes: Coccus (globular), Bacillus (rod) and Spirilla (spring or undulating)

Staining:> Preparation of bacterial smear: Smear, add a drop of water and air dry> Fixation – kills bacteria and adheres onto slide (heat fixation)> Always observe aseptic techniques and procedures, wear lab gowns, know procedure (amount and timing)> Application and types of stains:

1. Simple Stain2. Differential Stain – ex. Gram staining and Acid-fast staining

>apply the following in EXACTLY this order: Primary stain, Mordant (increases affinity to adhere to cell), Decolorizer, Secondary stain>GRAM STAINING: (Deep violet cells = +; Pink cells = -)

a. Prepare overnight cultureb. Prepare bacterial smearc. Flood smear with CRYSTAL VIOLET for 1 MINUTE. Rinse.d. Flood with GRAM’S IODINE for 1-2 MINUTES. Rinse.e. Decolorize with 95% ETHANOL for 15 SECONDS. Rinse IMMEDIATELY.f. Flood with SAFRANIN for 45 SECONDS. Rinse and blot dry.g. Observe at OIO.

>ACID-FAST STAINING: [Red-Pink bacteria = +; Blue Bacteria = - ] resistance to decolorization by acidsa. Prepare bacterial smearb. Cover smear with CARBOLFUSCHIN. Steam over boiling water for 8 minutes. DON’T ALLOW STAIN TO BOIL OFF. Cool.c. Decolorize with ACID-ALCOHOL for 15-20 SECONDS. Rinse.d. Counterstain with METHYLENE BLUE from 30 SECONDS. Rinse and blot dry.e. Observe at OIO.

3. Special stains> CAPSULE STAIN/ NEGATIVE (NIGROSIN) STAIN: (Halo around translucent-dense bacteria = + or with capsule)

a. On one end of glass slide, place one drop of 6% AQUEOUS GLUCOSE on each corner and emulsify a loopful of bacterial growth in it.b. Add a loopful of NIGROSIN/INDIA INK in suspension. Mix gently.c. Spread micture using edge of another slide. Air-dry and view under OIO.

> FLAGELLAR STAIN – accounts for the presence, number and arrangement of flagella around bacteria- To undergo this experiment, MOTILITY TEST AND HANG DROP TECHNIQUE must be positive

> SPORE STAINING (Schaeffer-Fulton Method): (Green mass inside Pink cell = with endospore or +)a. Prepare bacterial smear of OLD BACTERIA, air-dry and fix by heat.b. Cover smear with filter paper and flood with MALACHITE GREEN.c. Place above steam bath for 10 MINUTES. Make sure slide is always saturated with stain (don’t allow to boil). Cool and rinse.d. Counterstain with SAFRANIN for 1 MINUTE. Rinse, blot-dry and examine under OIO.

> GREGERSEN’S METHOD: (Viscous solution = Gram-negative; Watery suspension = Gram-positive)a. Place 1-2 DROPS OF 3% KOH on slide.b. Obatin loopful of overnight inoculum and mix with 3% KOH. Observe consistency using a wire loop.

III. Physiologic/Metabolic Tests> Used to test for presence of enzymes> See Nutritional and environmental requirements> See Nutritional consumption> Further production of certain compounds and/or substances> pH changes> Results: Color change in media, lysis/clearing, precipitate or compound formation, growth patterns

1. Nutrient Gelatin- test used to identify decomposition of gelatine- after pacing in refrigerator/ice bath, observe results- Positive = gel liquefaction (nutrient gelatin was consumed, broth remained liquid)

2. TSI (Triple Sugar Iron) Test-STAB AND STREAK

3. SC (Simmons-Citrate) Agar

-defined, enrichment medium that tests for an organism's ability to use citrate as a sole carbon source and ammonium ions as the sole nitrogen source.

- contains bromothymol blue, a pH indicator (green at pH below 6.9; blue at pH of 7.6 or greater)

4. Phenylalanine Agar

- add 4 DROPS of 10% FERRIC CHLORIDE SOLUTION after incubation for 24-48 hours and rotate tube (to loosen -growth)- Positive = Immediate disappearance of an intense green color

5. Casein Agar Plate

- Positive = clearing of previously milky-white medium indication hydrolysis of casein

6. Starch Agar Plate

- after incubation, flood surface with LUGOL’S IODINE or IKI- Positive – observe for clearing zones around are of inoculation (starch hydrolysis)

7. Nutrient Agar plus Tween 80 (NA + T80) Plate- Positive = white zone of precipitation around area of streak (indicates lipolytic activity)

8. Catalase Test- transfer inoculum on glass slide and add 1-2 DROPS HYDROGEN PEROXIDE- Positive = bubble formation on glass slide; Weak Positive = bubbles are seen under LPO only

9. Growth in Anaerobic Agar (Thioglycollate broth)- inoculate bacteria on thioglycollate broth (must be yellow; water bath if still pink until yellow color appears)a. Obligate aerobesb. Obligate anaerobesc. Facultative anaerobesd. Microaerophilese. Aerotolerant anaerobes

10. MR-VP (Methyl Red - Voges-Proskauer) Test

11. Indole Production from Tryptophan

- after incubation, add 2 drops of Kovac’s reagent and allow to stand without mixing- Positive = red colored ring on the interface (production of indole from tryptophan)

12. Oxidase Test ***a. Place a sterile filter paper on a glass slide and add 2-3 DROPS OF OXIDASE REAGENT to moisten.b. With an inoculating loop, transfer colony onto filter paper and spread to moistened regions.c. Development of LAVENDER/VIOLET COLOR within ONE (1) MINUTE indicates a positive test/result.

13. Urea Hydrolysis***

a. Inoculate overnight culture into Urea broth.b. Incubate for 24-48 hours.

c. Appearance of RED (from light orange) colored media indicates a positive test (urea was hydrolyzed).

******Dichotomous Key:

http://www.bmb.leeds.ac.uk/mbiology/ug/ugteach/newdental/Bacterial_id/neg/rods/aerobic/fastidious/index.htmlgram reaction(young culture)

+ + + + + + + + + + – – – – – –

shapecoccus

(clusters)coccus

(clusters)coccus

(chains)coccus

(tetrads)rod rod

irreg.rod

rod rod rod rod rod rod rod rodcoccus(pairs)

aerobic growth + + + + + + + – + + + + + + + +

anaerobic growth – + + + + – – + + – – – + + + –

endospores – – – – – – – + + + – – – – – –

motility(Motility Medium)

– – – – – + –+

or –+

or –+

or –+

or –+

or –– + + –

catalase reaction + + – – – + + – + + + + + + + +

benzidine reaction + + – – – + + – + + + + + + + +

oxidase reaction + – – – – – – –+

or –+

or –+ + – – + +

glucose fermentationto acid or to acid+gas

– + + + + – –+ (or

–)+ – – – + + + –

Glucose O/F Medium – O F F F O

Micrococcus X

Staphylococcus X

Streptococcus X

Lactococcus X

Enterococcus X

Leuconostoc X

Pediococcus X X

Aerococcus X

Lactobacillus X

Kurthia X

Arthrobacter X

Clostridium X

Bacillus X X

Alcaligenes X

Pseudomonas X

Klebsiella X

Shigella X

Salmonella X

Escherichia X

most otherenteric genera

X

Aeromonas X

Chromobacterium X

Neisseria X

Results for Identification of Unknown Bacterial Specimen:

I. Cultural Characteristics

Criteria Characteristic of Bacterial Unknown

Color of Colony White

Shape of Colony Circular

Margin of Colony Entire

Elevation of Colony Convex

Surface texture of colony Smooth and shiny

Shape of Cell Rod-shaped

II. Morphological Tests

Test Final Result/Observation Characteristic

Gram-staining Violet-colored cells GRAM POSITIVE

Gregersen’s Method Non-liquid, aqueous consistency GRAM POSITIVE

Acid-fast staining Blue cells NON ACID-FAST STAINING

Spore Stain No blue-green spores were stained NON-SPORE FORMING

Capsule/Negative(Nigrosin) Stain

No formation of halo NO CAPSULE

III. Physiologic/Metabolic tests

Test Final Result/Observation Characteristic

Nutrient GelatinLiquid consistency was maintained after being subject to low temperature

POSITIVE(+)

TSI (Triple Sugar Iron) Slant

SC (Simmons-Citrate) Agar

Phenylalanine Agar

Casein Starch Plate Clearing was observed at the streakPOSITIVE

(+)

Starch Agar Plate Clearing was observed around the streak POSITIVE(+)

Nutrient Agar plus Tween 80 (NA + T80)

PlateNo formation of white precipitate around streak NEGATIVE

(-)

Catalase Test Bubble formation was observed POSITIVE(+)

Growth in Anaerobic Agar (Thioglycollate

broth)Pink color found at the topmost part of the thioglycollate broth OBLIGATE AEROBE

MR-VP (Methyl Red - Voges-Proskauer) Test

Indole Production from TryptophanOxidase Test***

Urea hydrolysis***EMB (Eosin-

Methylene Blue) Agar