Exercise 8

What are biochemical tests used for in microbiology?

to identify microorganisms based on their enzymatic and metabolic activities

What are agar plate tests used to detect?

the presence of exoenzymes

• enzymes secreted outside the cell

What are examples of agar plate tests for exoenzymes?

• lipase test

• milk agar test

• starch agar test

What do tube media tests detect?

other metabolic processes inside the bacterial cell

What are examples of tube media tests?

• litmus milk

• sugar fermentation broth

• kligler’s iron agar

What is litmus milk test used for?

to test multiple metabolic activities of bacteria in milk-based media

What are the main components of litmus milk medium?

• lactose

• casein and other peptones

• litmus (pH indicator)

What processes does the litmus milk test detect?

• fermentation of lactose

• metabolism of proteins

• degradation of casein

• litmus reduction

How many types of reactions can be observed in the litmus milk test?

five

What are the litmus milk reactions?

• acid production

• alkaline production

• curd formation

• reduction (redox)

• proteolysis

What happens when bacteria ferment lactose in litmus milk?

acids are produced, which lower the pH of the medium

What color changes indicates lactose fermentation in litmus milk?

the litmus milk turns pink at acidic pH

What causes the pH drop during lactose fermentation in litmus milk?

the production of acidic byproducts from lactose metabolism

What happens when bacteria deaminate peptones in litmus milk?

ammonia is released into the solution

How does ammonia release affect the pH of the litmus milk medium?

it raises the pH, making the solution alkaline

What color change indicates peptone deamination in litmus milk?

the medium turns blue

Why is the blue color from peptone deamination in litmus milk often seen only at the top of the tube?

because peptone deamination is oxygen-dependent, so it occurs near the surface where oxygen is

When does litmus function as a pH indicator in litmus milk?

only when its in an oxidized redox state

What happens to the litmus indicator when it is reduced?

it loses its color

• medium turns white

What causes the litmus milk to turn white?

the bacterial culture creates a reducing environment, causing the litmus indicator to be reduced

Why does a colored band often remain at the top of the litmus milk tube?

because oxygen near the surface re-oxidizes the litmus indicator, restoring its color at the top

What enzyme do some bacteria produce that causes proteolysis in litmus milk?

caseinase

What happens when casein is degraded in litmus milk?

the milk turns clear

What is another name for proteolysis in litmus milk?

peptonization

What causes curding in litmus milk?

acid produced from lactose fermentation denatures milk proteins

How can curds appear in litmus milk?

• complete curd: milk fully solidifies

• incomplete curd: milk becomes lumpy or semi-solid

Does curding always occur when acid is produced?

no, acid production does not always lead to curding

What does penol red (PR) sugar fermentation broth test for?

the ability of an organism to ferment a specific sugar

What sugars can be included in PR broth?

• glucose

• lactose

• or mannitol

What indicators are used in PR broth?

• phenol red (pH indicator)

• durham tube (gas detection)

What happens if an organism ferments the sugar?

• phenol red turns yellow (acid production)

• gas may be produced (bubble in Durham tube)

What happens if an organism cannot ferment the sugar?

• peptone degradation releases ammonia

• phenol red changes to cerise color (alkaline reaction)

What metabolic activities does Kliglers Iron agar (KIA) test for?

• lactose fermentation

• glucose fermentation

• sulfur reduction

What are the carbohydrate sources in KIA?

• 1.0% lactose

• 0.1% glucose

What other ingredient provides ammonia and sulfur in KIA?

1.0% peptone (from cysteine metabolism)

What indicators are used in KIA?

• phenol red for pH changes

• iron (from ferric ammonium citrate) which reacts with H2S to form a black precipitate

What sugars are present in KIA and in what concentrations?

• glucose 0.1%

• lactose 1.0%

What happens when peptone is broken down in the presence of oxygen in KIA?

ammonia (NH3) is released, which can raise the pH

What occurs if both glucose and lactose are fermented in KIA?

acid products are produced in excess of NH3, turning the entire tube yellow

Why does the tube turn yellow when both sugars are fermented in KIA?

the acid from sugar fermentation lowers the pH, overwhelming any alkaline effect from NH3

How is gas production detected in KIA?

by lifting or cracking of the agar in the tube

What causes the lifting/cracking of the media in KIA tubes?

accumulation of fermentation gases produced by the bacteria

What happens when only glucose is fermented in KIA?

the NH3 produced from peptone breakdown exceeds the acid from glucose fermentation on the slant

Where does peptone breakdown occur in the KIA tube?

only in the presence of oxygen

• mainly at the slant

What is the pH of the slant during glucose-only fermentation in KIA?

neutral or alkaline, due to NH3 production from peptone deamination

What happens to the butt of the KIA tube during glucose-only fermentation?

it stays yellow because oxygen cannot penetrate, so acid from glucose fermentation remains

What is reversion in KIA?

when some lactose-fermenting bacteria produce weaker acids that can be oxidized to neutral end products, causing part of the slant to return to neutral/alkaline pH

Which bacteria commonly show reversion in KIA?

enterobacter aerogenes

What fermentation pathway leads to reversion?

2,3-butanediol fermentation pathway

Why do 2,3-butanediol products cause reversion?

they are less stable than mixed acid fermentation products (lactate, acetate, succinate, formate) and can be neutralized in aerobic environments

Is the lipase plate selective or differential?

differential media

What enzyme does the lipase plate test for?

lipase, which hydrolyzes fats into glycerol and fatty acids

What indicator is used in the lipase plate?

spirit blue

How is a positive result indicated on a lipase plate?

dark blue zone around the growth with no oily surfaces

What indicates a negative result on a lipase plate?

no color change around the growth

Is milk agar selective or differential?

differential media

What enzyme does milk agar test for?

caseinase, which hydrolyzes casein (milk protein) into amino acids

How does casein affect the appearance of milk?

casein gives milk its white color

How is a positive result indicated on milk agar?

clearing around the colony (loss of white color)

How is a negative result indicated on milk agar?

no clearing around the colony

Is starch agar selective or differential?

differential media

What enzyme does starch agar test for?

amylase, which breaks down starch into simple sugars

What indicator is used on starch agar?

iodine

How does iodine indicate the presence of starch?

iodine reacts with starch to produce a purple color

How is a positive result (starch hydrolysis) shown?

clear zone around the colony after adding iodine

How is a negative result with starch agar shown?

no clear zone

• medium turns purple where starch is present