Classification & Identification Of Bacteria

Question 1

Gram stain steps

Answer 1

Pour crystal violet (60 secs)

Wash off with water and flood with
iodine (60 secs)

Wash off with water and decolorise with 95% alcohol

Counterstain with safranin (60 secs) and wash off with water

Question 2

Cells that absorb the crystal violet and hold onto it-
⚫

Answer 2

Gram positive organisms stain blue or purple or black.

Question 3

If the cells do not absorb crystal violet, it is washed off by the alcohol, the cells will then absorb safranin or dilute carbol fuschin
⚫

Answer 3

Gram negative organisms stain pink or red

Question 4

Medically important bacteria use chemical and organic compds as energy source to produce ATP
⚫ Obligate intracellular organisms –not capable of metabolic pathways, so get ATP from host cells

Answer 4

Eg Chlamydia, Rickettsia

Question 5

⚫ How they deal with oxygen (in a spectrum)

Answer 5

⚫Molecular O2 forms radicals lethal to them, some are armed with enzymes that break them down.
⚫Obligate aerobes ⚫Facultative anaerobes ⚫ Microaerophilic ⚫Obligate anaerobes

Question 6

Classes of bacteria

Answer 6

Gram positive bacilli
⚫Gram positive cocci
⚫Gram negative bacilli (largest group)
⚫Gram negative cocci
⚫Others
⚫Mycobacteria –acid fast stain
⚫Spirochates –too small to be seen with light the microscope
⚫Mycoplasma –without cell
wall, only cell membrane

Question 7

Bacteria antigens

Answer 7

⚫ O (somatic) antigens to classify Gram negative bacilli
⚫ O antigen, H (flagella) antigen and Vi (capsular)
⚫ Over 2000 serotypes of Salmonella
⚫ M protein classifies S. pyogenes into >80
types
⚫ Cell wall carbohydrate antigen is used to group the Streptococci

Question 8

Basic characteristics to classify & identify bacteria

Answer 8

⚫ Colony shape/xteristic in culture
⚫ Haemolytic patterns (α, β, non)
⚫ pigmented
⚫ Specialised structures like flagella, spores, capsule
⚫ Biochemical reactions to ascertain presence of
metabolic activity
⚫ Fermentation (fermentative & non-fermentative bacilli)
⚫ Some characteristics
⚫ Motility

Question 9

Classify Escherichia coli from kingdom to species

Answer 9

Kingdom Prokaryote

Division Gracilicutes

Class Scotobacteria

Order Eubacteriales

Family Enterobacteriaceae

Genus Escherichia

Species coli

Question 10

Detecting & identifying bacteria

Answer 10

⚫ Culture
⚫ Cell free media – colonial morphology on
culture media
⚫ Microscopy
⚫ Stains -Gram stain (morphology), giemsa stain ricketsia –light microscopy
⚫ Wet prep - phase contrast or dark-field microscopy
⚫motility (hanging drop)
⚫Biochemical reactions, e. g,
⚫What sugars do they ferment,
⚫presence of respiratory enzyme

Question 11

Identifying features of bacteria’s

Answer 11

⚫ Colonial morphology
⚫ Oxidase reaction
⚫ Indole reaction
⚫ Urease production
⚫ Hydrogen sulphide production
⚫ Sugar fermentation/utilisation
⚫ Motile or nonmotile

⚫ Serology
⚫Agglutination, CFT,
fluorescent antibody test etc
⚫Antigen detection
⚫Latex agglutination
⚫Etc

Question 12

Detecting bacteria using DNA techniques

Answer 12

⚫ A probe is a section of DNA that is meant to adhere to its opposite strand of DNA
⚫ PCR – DNA can be amplified and then identified
⚫ has specificity and high speed capabilities
⚫ DNA sequence of a particular component of their
ribosomes
⚫ used as a tool to group bacteria together that are believed to have evolved from a common ancestor.
⚫ Their ribosomal RNA would be very similar (16s RNA)

Question 13

Classify based on how dangerous they are

Answer 13

⚫ Pathogens -are always likely to cause disease e. g, Salmonella typhi, Vibrio cholerae
⚫ Opportunistic -cause disease only when they have a special opportunity to gain entrance inside the body e g Bacteroides fragilis, Clostridium difficile, Pseudomonas aeruginosa
⚫ Invasive -proactively create portals of
entry - e.g, Salmonella enteritidis