Basic Microbial

Identification

Paige Shelley

Basic Microbial identification

What to ID and to what level?

Staining techniques

Basic biochemical tests

When to ID and to What level?

Level of ID is dependant on the type of organism and

the grade that the organism has been isolated from.

For example:

if a Gram +ve cocci is isolated within a Grade C

preparation area, it is unlikely to be fully identified.

However, if it is a result of an Action level excursion

then it will be identified to species level.

if a Gram –ve is isolated within a Grade C preparation

area, then this will go to full identification as they are

classed as ‘objectionable 'organisms.

Staining techniques

Organisms are firstly categorized by a staining

technique known as ‘Gram staining’.

A sample of a culture is heat fixed onto a microscope

slide before staining.

This technique uses a series of reagents to establish if

an organism has a peptidoglycan layer or not.

This is distinguished by a RED (Gram negative) or

PURPLE (Gram positive) stain when viewed under a

microscope (oil emersion).

Staining techniques.

Staining techniques

Gram staining:

Crystal Violet (flood slide for

1 minute)

Lugol’s iodine (Flood slide for 1 minute)

Acetone (Flood slide 3

seconds maximum)

Safranin (Flood slide for 1 minute)

Rinse slide with water

Tip- Put a small amount of acetone on some tissue/lint

free wipe and clean the back of the slide. This will mean

more clarity when viewing under the microscope.

Staining techniques

Gram staining

When to ID and to What level?

Is the organism a GPC?

Is the organism the result of an Action level?

Proceed to full identification

Report to Gram level only

Is the organism objectionable? (GNR, GNC, Yeast, Mould)

Proceed to full identification.

Yes No

Yes No Yes

Example of a

basic flow chart

in relation to a

Grade C isolate

and full ID

requirements.

When to ID and to what level?

Basic Biochemical Testing

Biochemical tests are used to confirm/deny a Gram

result

Used in addition to a Gram stain result to identify an

organism further e.g. to genus/species level

Basic Biochemical Tests: Catalase

Added to an organism that is a

Gram Positive Cocci, a positive

result will produce in gas. Thus

confirming the organism is a

Staph sp./Micrococcus sp.

Added to an organism that is a

Gram Negative Cocci, a negative

result will not produce gas. Thus,

confirming the organism is a

Gamella sp.

Basic Biochemical Tests: Oxidase

Add reagent to a sterile swab and

touch the desired colony. A purple

colour change within 10-30 seconds

is a positive reaction.

GNR +ve = Non-enterobacteriaceae

GNR –ve = both entero and non-

enterobacteriaceae

GNC +ve = Neisseria/Moraxella sp.

GNC –ve = perform catalase test

(cat +ve = Acientobacter sp. and cat

–ve= Gemella sp.)

Basic Biochemical Tests: Furazolidone disc

Used to distinguish between

GPC Micrococci and

Staphylococci.

Disc placed on a fresh sub

culture and incubated for 24

hours.

A halo of inhibition will confirm

a Staphylococcus sp.

If growth occurs right up to the

disc this confirms a

Micrococcus sp.

Basic Biochemical Tests: 3% KOH

Used to differentiate between a Gram positive and

Gram negative

A drop of the reagent is placed onto a clean

microscope slide.

Mixed colony with solution using a sterile loop for

60 seconds.

Positive= viscous stringy solution (like mucus)

Gram negative

Negative= No reaction

is a Gram positive

Microbial Identification- PRACTICAL

Please refer to hand out for Gram staining instructions,

we will talk through it and I will answer any questions.

I will be walking around to help everyone out.

Microscopy Tips and Tricks

Microscopy Tips and Tricks

Adjustable areas of the microscope include:

The stage

Light source

Lenses(including eye piece lens)

Course focusing wheel

Fine focusing wheel

Microscopy Tips and Tricks

Light Microscope:

The level of detail is down to the magnification of lenses.

They can range vastly depending on there use. For best

results when reading a Gram stain, an oil immersion lens

of 100x magnification (or higher) is required. This gives a

better resolution to the image upon viewing.

Microscopy Tips and Tricks

Maintenance:

Microscope should always have the dust cover on whilst

not in use. Lens specific wipes only to cleans lens glass.

Do not touch the lenses with hands/fingers. Only use

immersion oil on the specific lens designed for it. Always

clean oil immersion post use. Microscope should have a

service schedule to maintain its accuracy.

Microscopy Tips and Tricks- Reading Gram

stains

It is best practice within industry to ensure employees

receive eye tests (including colour blindness) before

performing colour subjective tests.

Variation in Gram stain reading is not uncommon.

Robust training and second checking of results is

common practice to ensure technician capability.

Microscopy Tips and Tricks- Reading Gram

stains

Gram Positive Cocci (GPC) Round cells with a purple coloured Gram stain result. They can be in

different formation.

Gram Negative Cocci (GNC) Round cells with a red/pink coloured Gram stain result. They can be in

different formation.

Coccus=single cell

Diplococcus=paired cells

Streptococus=string of cells

Staylococcus=look like “grapes”

Tetras= 4 cells (square)

Sarcina= Tetrad in 2 layers (cube)

Microscopy Tips and Tricks- Reading Gram

stains

GPC GNC

Microscopy Tips and Tricks- Reading Gram

stains

Microscopy Tips and Tricks- Reading Gram

stains

Gram Positive Rod (GPR) Rod shaped cells with a purple coloured Gram stain

result. Can be classified as Bacillus or Coryne.

Gram Negative Rod (GNR) Rod shaped cells with a red/pink coloured Gram stain

result.

Microscopy Tips and Tricks- Reading Gram

stains

Note: A dead give away for Bacillus is spore formation. A

cell that balloons at one end and/or contains a darker

internal circle means its is a spore forming Bacillus.

Microscopy Tips and Tricks- Reading Gram

stains

Gram Positive

Rod: Spore

forming bacillus

Microscopy Tips and Tricks- Reading Gram

stains

Gram Negative

Rod

Cellular Morphology

Objectionable Organisms

This categorisation is dependant on what type of

product/location the organisms is isolated from.

Generally organisms that have the ability to cause

‘harm’ or alter a product.

All pathogenic organisms are classed as

objectionable.

Companies are expected to risk assess the impact an

organism has on a patient/product and determine

objectionable status.

Objectionable Organisms

Gram Negative organisms

Mould species

Yeast species

Any spore forming organisms

Majority of pathogenic/opportunistic pathogenic

organisms are from the above families.

Objectionable Organisms

Burkholderia cepacia (Gram Negative rod) is generally

a non-harmful organism to healthy individuals. However

it has detrimental effects for individuals who have lung

related diseases (particularly cystic fibrosis). It is

therefore an objectionable organism.