MB Lab - Quiz 5

Bacterial Unknown Day #4

START hurray

All slants should be: ???? (for inoculation)

• Streaked AND stabbed

SIM Deep (how to inoculate)

• SINGLE stab into center of media

  • Make sure to enter and exit at same (because M = motility)

Simons Citrate Test (reagents + what test)

• Tests for: use citrate as SOLE carbon source

• NO REAGENTS TO ADD

Simmons Citrate (results)

• Positive = prussian blue

• Ngative = green color

Phenylalanine Deamination Test (Test for + reagent)

• Tests for

  • Presence of PHENYLALANASE (breaks down phenylalanine into phenylpyruvic acid (PPA))

• Reagent

  • 10% Ferric chloride

    • Why? Detects PPA with color change

Phenylalanine Deamination Test Results

• Results

  • Positive = DEEP GREEN

  • Negative = YELLOW (color of reagent aka NO color change)

#1 in picture = or reagent or inoculation

SIM Deep (test for + Reagents) (tests for 3 things) know reagents for each

• Tests for (and reagent that does it)

  • S = tests for H2S production

    • Reagent = Iron salt in media

  • I = detect INDOLE = presence of tryptophanase

    • Reagents: Chloroform and KOVAC’s reagent

      • Chloroform solubilizes the semi solid media

      • Makes permeable to Kovac’s

  • M = Motility = semi solid media

    • TTC (2,3,5- triphenyl tetrazolium chloride)

      • Colorless + soluble when oxidized; red when reduced

      • Makes it easy to see the results because the red color APPEARS along growth pattern

SIM Results + What reagents do you ACTUALLY ADD

• ACTUALLY ADD = chloroform + Kovac’s (chlorfomr to solubilize media)

• Positive

  • H2S = dark precipitate of iron sulfide

  • Tryptophanase/ Indole = red ring at top of media

  • Motility = growth radiating from red line

• Negative = none of those/ opposite of those

Day #5 Hydrolysis tests start

Tubes vs agar plates incubate temp and time

• Both incubate at 37

• Tube = 48 hours

• Agar plate = 24 hours

Urea Hydrolysis (test for + reagent)

• Tests for: urease presence

  • Hydrolyzes urea into ammonia + Co2

  • SO detects ammonia

• Reagent = Phenol red (already in media)

  • Detects ammonia, ammonia changes raise pH

    • Red = pH over 7

    • Yellow = pH under 7

Urea hydrolysis results

• Positive = red (fuchsia)

• Negative= yellow (orange)

Tryptone Hydrolysis (tests for + reagent)

• Tests for: Tryptophanase presence

  • Hydrolyzes tryotphan into INDOLE + PYRUVIC acid

• Reagent

  • Kovac’s reagent

    • Detects INDOLE (like the other test, SIM)

Tryptone Hydrolysis

• Positive= red ring at top

• Negative = no red ring

Fat Hydrolysis/ Spirit blue agar (test for + reagent)

• Tests for: lipase presence

  • Which can HYDROLYZE fats into glycerol + fatty acids

• Reagent (present in media)

  • Spirit blue agar = Contains LIPIDS

    • Need lipids to be hydrolyzed

Fat hydrolysis/spirit blue test

• Positive = Dark precipitate around inoculation (due to LOWERING of PH)

  • Lipid depletion around inoculation aka ZONE of hydrolysis

• Negative = NO dark precipitate

Casein Hydrolysis (Milk Agar) ( tests for + reagent)

• Tests for: Caseinase = hydrolyzes casein

  • Casein = predominant protein in milk (gives milk a white color)

• Resagent = NONE to add

  • Media = milk agar aka has casein/milk

Casein hydrolysis- results

• Positive = clear zone of hydrolysis around inoculation

• Negative = NO zone around growth, remains white

Starch Hydrolysis (tests and reagent)

• Tests for: Presence of amylase

  • (which can hydrolyze starch)

• Reagent

  • Media = Starch

  • Iodine = add GRAMS IODINE

    • Make thin layer across surface of agar

• Gorms complex/ binds to INTACT STARCh

  • turning NEGATIVE REULTS to blue/black

  • If it did, will stay clear beacue it wont bind

Starch Hydrolysis (results)

• Positive = clear zone around growth (zone of hydrolysis)

  • No color change

• Negative = NO clear area around growth

  • Media is STAINED brownish blue by starch iodine complex

IMViC test (what are the 4, and what is pos/neg for each category (2 categories)

• 4 physiological tests used to differentiate between ENTEROBACTERIAEASE or ENTERIC BACTERIA

• 4

  • Indole

  • Methyl red

  • Voges Proskaeur

  • Citrate

• E.coli = ++—

  • Indole + Methyl red = POSITIVE

• E. aerogenes = —++

  • VP + Citrate = POSITIVE

What does second I stand for in IMViC

• Just for pronunciation

API 20E Identification for Enterobacteriaeae and other Gram negative

Start

What is the API 20E system

• Minaturized version of convential l TUBE TESTS for bacterial unknowns

  • Gram negative rod that is member of enterobacteriaceae

Inoculation of test strip (important steps)

• Fill EACH DEPRESSION with a drop of so of whater

  • DO NOT OVERFLOW

  • Used to create humid environment

• Create suspension of bacterial unknown

  • MAKE SURE TO MAKE TURBID

• Use sterile Pasteur pipet w/ rubber bulb

Tube vs Cupule

• Tube = lower part

• Cupule = part open to the air

Which tests to NOT fill suspenion up to cupule (aka which nee dot be anaerobic) - 5 add MINERAL oil

1. ADH

2. LDC

3. ODC

4. H2S

5. URE

Think —- HALO U

TDA Reagent + result

• Reagent = add ferric chloride

  • Positive = brown-red

  • Negative = Yellow

VP Reagent + result

• Reagent = Barritt’s (a-naphthol + KOH)

• Positive = pink or red (appears within 10 minutes)

IND (reagent and tests)

• Kovac’s reagent, NO CHLOROFORM NEEDED??

• Positive= red ring within 2 minutes

NIT

• Reagents = sulfanilic acid + NN, dimethyl 1 napthylamine

• Positive = red color

• Negative = ADD ZINC to test

UV Light Lethal effects start

START ya

How to swab plate

• TRYINT TO MAKE a fully inoculated plate

• Swab with cotton swab in 3 diff directiosn in zig zag

What is UV light + total range

• electromagnetic energy

• 1nm to 400nm

3 groups of UV light

UV A, B, C

UV -A

• 315-400 nm

• LONGEST

UV-B

• 280-315nm

UV-C

• 100-280nm

• MOST DANGEORUS TO BACTERIA

Most dangerous wavelengths for bacteria (3 important ones)

• 265= MOST DAMAGING to DNA (emitted by our UV lamps)

  • WHy?

    • Most efficient for cell penetraiton and pyrimidien dimers

• 280nm = protein concentration determined

  • Proteins absorb UV light at this wavelength MAXIMALLY

• 260 = wavelength that DNA and RNA concentration is determined

  • High absorbance from DNA bases

How does UV light cause bacterial death

• UV light causes DNA damage in form of PYRIMIDINE DIMERS

  • Thymine dimer = most common

  • COVALENT bonds between thymine ===== distort DNA helix + blocks replication genreally because cant read DNA

• CANNOT be repaired by organisms DNA repair mechanism

UV Lights distance from plate

5 inches

Which bacteria = ENDOSPORE FORMER and what does this do

• Bacillus subtills = ENDOSPORE FORMER

  • Can withstand longer exposures

• Will have some growth rather than none

  • E.coli + S.epidermidis = no growth at extended exposure (2.5-5)

Once conditions return to normal, how does b.subitlis function

• During incubation/ normal ocndtion, spore can GERMINANTE and new VEGETATIVE CELL will grow