Lab 1 - Bacterial Isolation

slide fixing, gram staining and identification, colony description and different medias, catalase test, oxidase test, motility test, oxidation/fermentation test, API tests, common issues

What is the process of identifying a bacteria (6 different steps)?

1. specimen taken

2. bacteria cultured

3. preliminary identification

4. further identification

5. antibiotic susceptibility

6. final report

What tests are carried out for preliminary identification of a bacteria (3)?

• colony morphology / growth

• gram stain

• oxidase and catalase tests

What tests are carried out during further identification of a bacteria (after plate growth and colony morphology / gram stain) (2)?

• motility test

• oxidation / fermentation tests

What type of media is blood agar?

general purpose media

What type of media is MacConkey?

selective / differential media

What is shown in this image?

plate streaking method for microbiology

How can blood agar be a differential media?

detects haemolytic activity

How is MacConkey agar a differential media (2)?

• lactose fermenters - red colonies

• non-lactose fermenters - colourless colonies

How is MacConkey agar a selective media (3)?

• bile salts and crystal violet dye

• gram positive growth inhibited

• non-enteric gram negative growth inhibited

What should you look for when looking at a bacterial culture grown on agar (4)?

• is there growth ?

• is the culture pure ?

• type of colony - shape, elevation, margin

• type of haemolysis (blood agar)

What type of blood agar haemolysis is shown in this image?

alpha haemolysis

What type of blood agar haemolysis is shown in this image?

beta haemolysis

What type of blood agar haemolysis is shown in this image?

gamma haemolysis

What is alpha haemolysis and how is it identified (2)?

• incomplete lysis of red blood cells

• green area around colony growth

What is beta haemolysis and how is it identified (2)?

• complete lysis of red blood cells

• clear area around colony growth

What is gamma haemolysis and how is it identified?

growth with no blood cell lysis

What are the steps to perform a gram stain (8)?

• fix slide

• crystal violet

• wash with water

• iodine

• decolourise with acetone

• wash with water

• counter stain with basic fuchsine

• wash with water and blot dry

What different chemicals are used in a gram stain (4)?

• methyl violet

• iodine

• acetone

• basic fuchsine

What type of bacteria are purple after a gram stain?

gram positive

What type of bacteria are pink after a gram stain?

gram negative

What type of bacteria is shown in this image (basic description - not actual name) ?

gram negative rods (image of E.coli)

What type of bacteria is shown in this image (basic description - not actual name) ?

gram negative cocci

What type of bacteria is shown in this image (basic description - not actual name) ?

gram positive rods (image of Listeria)

What type of bacteria is shown in this image (basic description - not actual name) ?

gram positive cocci (image of Staphylococcus)

What type of bacteria is shown in this image (basic description - not actual name) ?

gram positive cocci (image of Streptococcus)

What is detected by a catalase test?

presence of catalase enzyme

How is a catalase test carried out and what is a positive result (2)?

• colony dipped into hydrogen peroxide

• positive result - bubbles of oxygen given off

What is detected by an oxidase test?

presence of cytochrome oxidase

How is an oxidase test carried out and what is a positive result (2)?

• drop oxidase reagent onto a swab and touch to colony

• positive result - rapid appearance of purple colour on swab

Which tube indicates a negative motility test result?

middle tube

Which tube indicates a positive motility test result?

furthest right tube

Which set of tubes indicate the bacteria tested is fermentative?

furthest left tubes

Which set of tubes indicate the bacteria tested is oxidative?

middle tubes

Which set of tubes indicate the bacteria tested is neither fermentative nor oxidative?

furthest right tubes

What do API strips allow identification of?

bacterial genus and species

What API strip should be used for a gram negative bacteria?

API 20E

What API strip should be used for a gram positive bacteria?

API Coryne

What are some of the issues that can be encountered when gram staining (4)?

• smear is too thick

• mixed bacterial cultures (different shapes / colours)

• not washed properly between steps

• issues with decolourisation

What are some possible issues encountered when using API strips (4)?

• correct strip used?

• was the culture pure?

• correctly examined / recorded?

• were bubbles present?

What are some possible issues encountered when carrying out oxidation/fermentation and motility tests?

were tubes innoculated with enough bacteria?

What is a possible issue when carrying out oxidation/fermentation tests?

was a cm of oil placed on top of one tube (anaerobic conditions)?

What are these images of?

API test strips

What are the steps to fix a bacteria sample onto a slide for gram staining / microscope examination (4)?

1. place loop of distilled water onto clean slide

2. using disposable loop pick up minute quantity of colony

3. make emulsion of bacteria into the water and spread thinly on slide

4. fix by placing onto a heating bench until dry