Microbiology Lab Practical Review Part 2

Kirby-Bauer disc diffusion method

will be used to determine the sensitivity of E.coli and S.aureus to different antibiotics

• Compare to standards to determine sensitive, intermediate, or resistant

Sensitivity

is determined by the presence of a zone of inhibition (an area around an antibiotic disc that shows no growth)

How to determine sensitivity or resistance using the Kirby-Bauer disc diffusion assay

grow a standardized lawn of your test organism on Mueller–Hinton agar, place antibiotic discs, incubate, measure the zone of inhibition (mm) around each disc, and compare each diameter to published breakpoints

CFU/mL Calculation

CFU/mL = (Number of colonies × Dilution factor) / Volume plated
(e.g., 150 colonies × 10³ / 0.1 mL = 1.5 × 10⁶ CFU/mL)

Transformation

the uptake of either linear or plasmid DNA by a bacterial cell from its surroundings

Conjugation

transfer of DNA via direct cell-to-cell contact

Complementation

restoring wild type function to a mutant by reintroducing the wild type gene on a plasmid

Competent Cell

cells that are capable of transformation (cells can be made competent in a variety of ways)

Instruments used in the experiment

• Micropipettes, gel electrophoresis apparatus, water bath/heat block, centrifuge.

How to determine the size of unknown DNA band using a ladder

Sample bands are compared to a DNA ladder to estimate size of fragments (with known sizes)

Structure of the lac operon

a cluster of genes and regulatory sequences that control the metabolism of lactose. It includes three structural genes and three major regulatory regions

• lacI — CAP site — Promoter (P) — Operator (O) — lacZ — lacY — lacA

lac genes

lacZ, lacY, lacA

Role of reagents in Beta-galactosidase assay

• ONPG: ortho-nitrophenyl-β-galactopyranoside

• Cleaved by β-galactosidase – yields yellow compound (ONP)

• Reagents measure enzyme activity

lacZ

• Encodes β-galactosidase

• Function: breaks lactose → glucose + galactose; also converts lactose → allolactose (the inducer

lacY

• Encodes lactose permease

• Function: transports lactose into the cell

lacA

• Encodes thiogalactoside transacetylase

• Function: detoxification (not essential for lactose metabolism)

Promoter

• RNA polymerase binding site

• Required to start transcription of lacZYA

Operator

• Binding site for the lac repressor (LacI protein)

• Located just downstream of the promoter

• Repressor binding blocks transcription

lacl gene (repressor gene)

• Encodes the LacI repressor

• Located upstream, has its own promoter (not part of the operon’s mRNA)

• LacI binds operator unless lactose/allolactose is present

CAP binding site / cAMP–CAP complex

• Located upstream of the promoter

• When glucose is low → high cAMP → cAMP + CAP bind to site, increasing transcription

• When glucose is high → CAP does not bind → low transcription even if lactose is present (catabolite repression)

How to determine the presence of sulfur-reducing bacteria?

• Many sulfur-reducing bacteria are obligate anaerobes – not S.
  oneidensis

• S. oneidensis will respire using oxygen if it’s available, anaerobic environment forces it to use sulfur

• Clearing zones in sulfur plate

What coliform bacteria and their role

• Gram-negative rods that ferment lactose → indicator of fecal contamination.

• Aerobic or facultative anaerobes that grow

How EMB media is selective for coliforms

inhibits Gram-positive

How EMB media is differential for Coliforms

distinguishing lactose fermentation

Appearance of coliform bacteria on EMB agar

• Lactose fermentation produces acids, which lower the pH and allows dye absorption by the colonies turning purple-black.

• Lactose non-fermenters may increase the pH by deamination of proteins. The dye is not absorbed. The colonies will be colorless

α-hemolysis (alpha)

sometimes called green hemolysis because of the color change in the agar. This is a partial hemolysis. Red blood cells are only partially broken down

β-hemolysis

sometimes called complete hemolysis, is a complete lysis of red blood cells in the media around and under the colonies; the area appears lightened and transparent

γ-hemolysis

is when an organism does not induce hemolysis; the agar under and around the colony is unchanged

How to interpret throat culture results?

identifies streptococcal species

How the agar overlay assay works to identify antibiotic producing colonies

detects colonies that produce antibiotics by looking for zones where they inhibit the growth of a test organism

• Plate bacteria

• Overlay with sensitive indicator strain

• Clear zones = colonies producing antibiotics

Catalase

• Tests for Gram-positive only

• Most O2 respiring bacteria have catalase to protect them from peroxides produced during respiration

Positive and Negative Catalase

• Positive = Bubble formation

• Negative = No bubbles

Oxidase

• Perform on Gram-negative bacteria

• Some bacteria have cytochrome c oxidase as a component of their electron transport chain

• Dimethylphenylenediamine (DMPD) - artificial electron donor when cytochrome c is present

Positive and Negative Oxidase

• Positive = turns red in the presence of cytochrome c within 10 seconds

• Negative = No red color in 10 seconds

Gelatin

Detects proteolytic enzymes secreted by bacteria

Positive and Negative Gelatin

• Positive = Liquid

• Negative = Solid (hydrolyzed)

Glucose-iron Agar

• will be tested by the Phenol red in the agar (red color in neutral pH)

• Amino acid hydrolysis – produces H2S

  • H2S reacts with iron salts in the media to produce FeS (a black precipitate).

  • O2 requirement can be tested for by where the microbes grow.

    • Surface only – strict aerobes

    • Yellow/black in the agar column (bottom) - Facultative anaerobes

Positive and Negative Glucose-iron

• Positive = Yellow color from fermentation, cracks from gas production, black precipitate

• Negative = Red color, no cracks in agar

Starch

• This will test for amylase production.

• Amylase hydrolyzes starch and breaks it down into a more useable form

Positive and Negative Starch

• Positive = Crack formation, yellow color, black color

• Negative = Red color, no change, no cracks

Lactose and Sucrose

• This test also uses phenol red to detect fermentation products.

  • Acidic products – Yellow color

  • Gas production - Gas in the Durham tube

Positive and Negative Lactose and Sucrose

• Positive = Yellow color, gas production

• Negative = Red color, no gas production