Classification and Identification

Question 1

What are the types of microorganisms?
What form do they exist in?
Which have cell walls?

Answer 1

bacteria, algae, yeast, moulds, protozoa
virus - generally not included as they re ‘not-living’

can exist as
unicellular, multicellular or cell clusters

cell walls in
bacteria, algae, yeast, moulds - cell walls can be identified using gram stain method

Question 2

What is the difference between gram positive and gram negative bacteria?

Answer 2

gram positive

• have thick peptidoglycan cell wall = several layers
• plasma membrane = cytoplasmic membrane

gram negative

• outer membrane = lipopolysaccharide and proteins
• lipoproteins
• periplasmic membrane = between inner and outer membrane
• thin peptidoglycan cell wall
• plasma membrane = cytoplasmic membrane

Question 3

What is the method for gram staining?

Answer 3

1 - flood the smear with crystal methyl violet = primary stain
2 - flood with iodine = forms complex with crystal violet
3 - decolourise with 95% ethyl alcohol or acetone = dehydrates peptidoglycan, destains and allows differentiation between gram + and - = decolourising agent
5 - flood with safranin = counter stain

gram positive - retains crystal violet purple stain due to thick cell wall

gram negative - retains pink/red safranin stain due to crystal violet stain being removed

Question 4

How do genetic probes identify bacteria?

Answer 4

used to identify species specific DNA and RNA sequences
used to locate specific genes
- have a base sequence complementary to the base sequence of the target allele
- labelled with a compound to identify where it goes

compounds

• fluorescence dye
• peroxidase

Question 5

What is the difference between macroscopic and microscopic identification?

Answer 5

macroscopic 
- appearance of colony size, shape, colour
- pigment = colour 
- speed of growth
morphology 
- form - shape, texture 
- elevation - flat, raised 

microscopic

• cell shape and size = cocci, bacillus, spiral, vibrio
• gram stain
• acid fast reaction
• endospore? capsule? granule?

Question 6

What are the types of bacterial identification?

Answer 6

colony morphology or gram stain
biochemical test
- oxidase, catalase, urease, coagulase 
acid fast stain
spores
growth character - oxygen, temperature, pH
motility 
antigenic properties
- molecular or genetic characteristics

Question 7

How can bacteria be cultured? What are they cultured in?

Answer 7

inoculum is introduced to a medium
inoculum - sample, medium - collection of nutrients

microorganisms that are grown from an inoculum are called a culture

culture can be grown in
broth - liquid
agar - solid

Question 8

What should a culture media contain?

Answer 8

source of protein
pH control
defined salt concentration

Question 9

What are the types of agar and what do they contain?

Answer 9

agar

• is a complex polysaccharide derived from the cell walls of red algae
• most microbes cannot digest agar

nutrient agar
- peptone, beef extract, NaCl, agar

tryptone soya agar
- casein enzymichydrolysate, papaicdigest of soyabeanmeal, NaCl, methylumbelliferyl-β-D-glucuronideand agar

Question 10

What is a progenitor?

Answer 10

may be either a single cell or a group of related cells
is termed a colony forming unit
- colony is an aggregate of cells derived from a single progenitor cell

Question 11

What are the types of culture media?

Answer 11

defined media
- exact chemical composition is known, synthetic

complex media

• contains nutrients that are released by the partial digestion of beef, yeast and soy
• variety of growth factors means it supports a wider range of microbes = used to grow organisms whose exact nutritional needs are not known

selective media
- contains substances that either favour the growth of a specific microbe or inhibit the growth of unwanted microbes

differential media

• used to distinguish one microbe from another = grow microbes on the same media
• uses biochemical interactions of microbes growing in the presence of specific nutrients/indictors = positive or negative results

anaerobic media

• used to grow anaerobic/facultative microbes
• contain components that chemically combine with and remove oxygen from the media

transport media
- used to move specimen from one location to another

media can be selective and differential

Question 12

How are microscopes used to identify bacteria?

Answer 12

wavelength of radiation

• radiation differs in wavelength
• electrons move as waves

magnification

• increases size of the objects
• occurs when the beam of radiation refracts as it passes through a lens

resolution
- ability to distinguish two objects that are close together

contrast

• refers to differences in intensity between two objects or between an object and its background
• important in determining resolution

Question 13

How does electron microscopy work? What are the different types?

Answer 13

must work in a vacuum
uses electrons of wavelengths between 0.01 nm – 0.001 nm = makes resolving and magnification power stronger

types

• transmission electron microscopes = specimen must be thin
• scanning electrons microscopes

Question 14

How does light microscopy work?

Answer 14

uses light = visible, UV
uses a series of lens for magnification

oil immersion increases resolution and magnification
- add oil to the area being observed

Question 15

How does staining allow identification? What are the types of stain?

Answer 15

must microbes are colourless
staining increases contrast and resolution
- necessary for light and electron microscopy

simple stain

• composed of basic dye
• involves only soaking the sample then washing off

differential stain
- uses more than one dye

Question 16

How are Analytical Profile Index Strips used to identify bacteria?

Answer 16

API 20E
used to identify gram negative bacilli (rod)

dehydrated substances are stored in the wells
- detect enzymatic activity
contains miniature biochemical tests

Question 17

How does acid fast stain work?

Answer 17

Stains Mycobacteria and Nocardia = bacteria

• flood slide with carbol fuchsin and heat
• the heat drives the stain through the waxy wall and into the cell
• decolourize with HCl and alcohol
• counterstain with methyleneblue

positive - red/ purple
negative - blue

Question 18

How does endospore stain work?

Answer 18

conventional stains will not work, as the spore coat is practically impermeable to all chemicals

• Schaeffer-Fulton endospore stain uses heat to drive the primary stain, malachite green, into endospore
• after cooling, the is slide decolourized with water and counterstained with safranin

positive - green
negative - pink