Practical 1: Differentiation of Bacteria & Induction of Bacterial Gene Expression During Infection

Practical Exam Flashcards pt 1

Dichotomous Scheme

Identify bacteria using phenotypic characteristics

K-OH Test (potassium hydroxide)

Identify whether bacterium is gram-positive or gram-negative.

• Quickly lyses gram-negative cells (weaker cell envelope) and DNA observed as slimy substance

K-OH Test Procedure

1. Place colony of bacterial isolate on microscope slide with inoculate loop

2. Place drop of 3% of K-OH onto bacterial colony

3. Mix bacteria and observe consistency

   1. Viscous and ‘stringy’: gram-negative

   2. Not viscous: gram-negative

Cell Morphology (Microscopy)

Identify cell shape using a microscope

Oxidative-Fermentative (O-F) Test

Tests if bacteria (particularly gram-negative) can grow in the presence or absence of oxygen

O-F Test Procedure

1. Two growth medium test tubes are inoculated with bacteria

   1. pH indicator in medium: turns medium yellow when bacteria acidifies

2. One tube is sealed with paraffin (prevents gas exchange)

3. Tubes incubated for 24-48 hours

Oxidase Test

Determines if bacterium possesses certain cytochrome c oxidases which utilizes oxygen for energy production via electron transport chain.

• Uses N,N’,N’-tetramethyl-p-phenylenediamine (TMPD) as redox indicator

• Reagent = dark blue when oxidized by giving electrons to cytochrome C oxidase

• Colorless when initial reduced state

Oxidase Test Procedure

• Place colony on DrySlide card with inoculating loop

• Purple/dark blue immediately = positive oxidase

• Pink or delated reactions = negative oxidase

Catalase Production Test

Used to help further distinguish gram-positive bacteria

• E.g. Gram-pos (K-OH negative), rod shaped bacteria, catalase positive could be bacillus, listeria, brevibacteria, or corynebacteria

Catalase Production Explanation

• aerobic and anaerobic bacteria produce hydrogen peroxide as oxidative end-product of aerobic breakdown of sugars

• hydrogen peroxide accumulates = highly toxic

• Bacteria protect by producing catalase to convert toxic hydrogen peroxide to wet er and oxygen

Catalase Reaction

2 H2O2 = 2 H2O + O2

• CATALASE POSITIVE: hydrogen peroxide added to bacterial cells → bubbles of oxygen

asr gene

• highly acid-inducible

• up-regulated during salmonella regulation

• encodes small basic periplasmic protein of 102 amino acids (10.6 kDa)

  • required for growth of bacterium under acidic conditions

• highly expressed during growth at 4.5 and 4.0 pH

Northern Blot

technique used to visualize RNA (asr mRNA) as a ‘band’ on a membrane

Green Florescent Protein (GFP)

Visualizes when gene is expressed

1. E.coli cells transformed with plasmid carrying genetic reporter pPasr::gap

2. Plasmid carries genetic fusion between acid-responsive promoter of asr gene, controls expression, and gene encoding GFP

3. transcription initiated from asr promoter in respinse to acidic conditions → mRNA transcribed from downstream gfp gene → make bacteria florescence when exposed to light

pPasr::gfp Nomenclature

• p: indicates genetic information is present on plasmid

• P: indicates only promoter sequence of asr gene present in plasmid

• :: indicated pPasr is fused to gfp

• gfp: indicates pretense of complete gfp gene

ori - plasmid feature

origin of replication: required for maintenance and replication of plasmid

bla - plasmid features

beta lactase-encoding gene: provides bacteria with ampicillin resistance; required to select for cells that carry plasmid and for plasmid maintenance

pPasr::gfp Procedure

1. Three strains of E.coli

   1. E.coli DH5a ‘N’: negative control; no gfp expression

   2. E.coli DH5a (pPrpsM::gfp) ‘P’: positive control; constitutive gfp expression

   3. E.coli DH5a (pPasr::gfp) ‘E’: Experimental culture