Medical Microbiology I - Lecture3

MEDICAL MICROBIOLOGY I

Lecture 3

Culturing of bacteria anaerobically, on

special agar and enriched agar

Anaerobic Culture

Anaerobic bacteria need special media for

growth because they need low oxygen

content, reduced oxidation-reduction potential

and extra nutrientsand extra nutrients

Media for anaerobes may have to be

supplemented with nutrients like hemin and

vitamin K

Boiling the medium serves to expel any

dissolved oxygen

Anaerobic Culture

Addition of 1% glucose, 0.1% thioglycollate,

0.1% ascorbic acid, 0.05% cysteine or red hot

iron filings can render a medium reduced

Robertson cooked meat that is commonly Robertson cooked meat that is commonly

used to grow Clostridium species.

Medium contain a 2.5 cm column of bullock

heart meat and 15 mL of nutrient broth

Anaerobic Culture

Before use the medium must be boiled in

water bath to expel any dissolved oxygen and

then sealed with sterile liquid paraffin

Methylene blue or resazurin is an oxidation- Methylene blue or resazurin is an oxidation-

reduction potential indicator that is

incorporated in the thioglycollate medium

Under reduced condition, methylene blue is

colourless

Gas-Pak System

Generating an anaerobic atmosphere within an

anaerobic jar

1. Inoculated media are placed in an anaerobic jar

along with a catalyst (consists of a disposable along with a catalyst (consists of a disposable

H2 and CO2 envelope) and an indicator strip

(blue)

2. A Gas-Pak envelope is activated by cutting off a

top corner of the envelope and placed inside

the jar

3. 10 mL of water is added into the jar

Gas-Pak System

5. The jar is sealed and placed in an aerobic

incubator

6. An anaerobic atmosphere should be attained

within 2 hours after sealing the jar.within 2 hours after sealing the jar.

7. If an anaerobic atmosphere has been

attained, the strip will turn white and

moisture will accumulate on the side of the

jar

Gas-Pak System

H2 and CO2 are generated in the following

manner

Citric acid and sodium bicarbonate react to

form waterform water

Sodium hydride reacts with water to form

sodium borohydrate and hydrogen

The palladium-coated catalysts reacts with

hydrogen and any remaining oxygen to form

water

Enriched Media

Addition of extra nutrients in the form of

blood, serum, egg yolk etc. to basal medium

makes them enriched media

Enriched media are used to grow nutritionally Enriched media are used to grow nutritionally

exacting (fastidious) bacteria

Blood agar, chocolate agar, Loefflers serum

slope etc. are few of the enriched media

Blood Agar

A general purpose medium used to cultivate

nutritionally demanding microorganisms and to

differentiate between those that lyse red blood

cells (haemolytic) and those that do notcells (haemolytic) and those that do not

It is especially useful to distinguish streptococci

based on their haemolytic properties

Bacteria growing on BA produce a wide variety

of products that affect the integrity of red

blood cells contained in the medium

Blood Agar

Streptococcus pyrogenes produce streptolysin

P and S, both of which are capable of

completely lysing red blood cells

Streptolysin O is oxygen-labile, where as Streptolysin O is oxygen-labile, where as

streptolysin S in oxygen-stable

Some streptococci are able to partially lyse the

red blood cells, resulting in greenish or

brownish discolouration around the colony -

alpha haemolysis

Blood Agar

Beta haemolysis appears as a clear or

colourless zone surrounding the colony

Species that fail to produce visible effects on

red blood cells are said to be gamma red blood cells are said to be gamma

haemolytic or non-haemolytic

Haemolysis is due to the release of enzymes

by microorganisms

Blood Agar

Selective and Enrichment Media

Designed to inhibit unwanted commensal or

contaminating bacteria and help to recover

pathogen from a mixture of bacteria

While selective media are agar based, While selective media are agar based,

enrichment media are liquid in consistency.

Both these media serve the same purpose

Any agar media can be made selective by

addition of certain inhibitory agents that DO

NOT affect the pathogen

Selective Media

Various approaches to make a medium

selective include:

1. addition of antibiotics

2. addition of dyes2. addition of dyes

3. addition of chemicals

4. alteration of pH

5. combination of some of these

Enrichment Media

A medium of known composition and specific

conditions of incubation which favours the

growth of a particular type or species of

bacteriumbacterium

Liquid media that also serves to inhibit

commensals in the clinical specimen

Selenite F broth, tetrathionate broth and

alkaline peptone water are used to recover

pathogens from faecal specimens

Chocolate Agar

A non-selective, enriched growth medium

It is a variant of blood agar

It contains red blood cells which have been lysed

by heating very slowly to 56Cby heating very slowly to 56C

Used for growing fastidious (fussy) respiratory

bacteria such as Haemophilus influenza

These bacteria need growth factors, like NAD

and hematin which are inside red blood cells,

thus a prerequisite to growth is lysis of the red

blood cells

Chocolate Agar

Differential Media or Indicator

Media

Certain media are designed in such a way that

different bacteria can be recognized on the

basis of their colony colour

Various approaches include incorporation of Various approaches include incorporation of

dyes, metabolic substrates etc. so that those

bacteria that utilize them appear as differently

coloured colonies

Examples: MacConkey agar, CLED agar, TCBS

agar, XLD agar etc.

MacConkey Agar

Grow Gram-negative bacteria and stain them

for lactose fermentation

It contains bile salts, crystal violet dye, neutral

red dye, lactose and peptonered dye, lactose and peptone

The bile salt will inhibit most Gram negative

bacteria, EXCEPT Enterococcus and some

species of Staphylococcus e.g. S. aureus

MacConkey Agar

MacConkey Agar

Crystal violet dye will inhibit certain Gram

positive bacteria

The addition of neutral red dye stains the

microbes fermenting lactosemicrobes fermenting lactose

Acting as a visual pH indicator, the agar

distinguishes those Gram negative bacteria

that can ferment the sugar lactose (Lac+) from

those that cannot (Lac-)

MacConkey Agar

By utilizing the lactose available in the

medium, Lac+ bacteria such as Escherichia coli,

Enterobacter and Klebsiella will produce acid

This acid will lower the pH of the agar below This acid will lower the pH of the agar below

6.8 and results in the appearance of red / pink

colonies

Non-lactose fermenting bacteria such as

Salmonella, Proteus species and Shigella

cannot utilise lactose and will use peptone

instead.

MacConkey Agar

This forms ammonia, which raises the pH of

the agar and leads to the formation of white /

colourless colonies

Some organisms ferment lactose slowly or Some organisms ferment lactose slowly or

weakly and are sometimes put in their own

category.

These include Serratia and Citrobacter

Eosin Methylene Blue (EMB) Agar

A selective stain for Gram negative bacteria

It is a blend of two stains: eosin and

methylene blue (6:1)

Inhibits the growth of Gram positive bacteria Inhibits the growth of Gram positive bacteria

Provides a colour indicator distinguishing

between organisms that ferment lactose (e.g.

E. coli) and those that do not (e.g. Salmonella,

Shigella)

Eosin Methylene Blue (EMB) Agar

Eosin Methylene Blue (EMB) Agar

Lactose fermentation produces acids which

lower the pH

This encourages dye absorption by the

colonies, display nucleated colonies which colonies, display nucleated colonies which

are now coloured purple-black in the centers

Lactose non-fermenters may increase the pH

by deamination of proteins

This ensures that the dye is not absorbed

Eosin Methylene Blue (EMB)

On EMB if E. coli is grown it will give a

distinctive metallic green sheen (due to the

metachromatic properties of the dyes, E. coli

movement using flagella, and strong acid end-movement using flagella, and strong acid end-

products of fermentation)

Some species of Citrobacter and Enterobacter

will also react this way to EMB

Mannitol Salt Agar (MSA)

It contains a high concentration (7.5 - 10%)

of salt (NaCl) making it selective for

staphylococci and Micrococcaeae

Since this level of NaCl is inhibitory to most Since this level of NaCl is inhibitory to most

other bacteria, it is also a differential medium

This medium contains mannitol and the

indicator phenol red

It is a transparent, reddish pink agar

Mannitol Salt Agar (MSA)

Mannitol Salt Agar (MSA)

Coagulase positive staphylococci produce

yellow colonies with yellow zones (on the

medium itself)

Coagulase negative staphylococci produce Coagulase negative staphylococci produce

small pink or red colonies with no colour

change to the medium

The medium is used for the selective isolation

of presumptive pathogen (pp) staphylococci

Thayer-Martin Agar

A Mueller-Hinton agar with 5% chocolate blood

(lysed blood) and antibiotics

It is used for culturing and primarily isolating

pathogenic Neisseria bacteria including N. pathogenic Neisseria bacteria including N.

gonorrhoeae and N. meningitidis as the medium

inhibits the growth of most other

microorganisms

When growing N. meningitidis, usually will start

be inoculating a normally sterile body fluid

(blood or CSF) so a plain chocolate agar is used

Thayer-Martin Agar

Mueller-Hinton Agar

Mueller-Hinton agar is a microbiological

growth medium that is commonly used for

antibiotic susceptibility testing.

It is also used to isolate and maintain Neisseria It is also used to isolate and maintain Neisseria

and Moraxella species

Mueller Hinton Agar

Bile Esculin Agar (BEA)

Selective differential agar used to isolate and

identify members of the genus Enterococcus,

also known as group D streptococci

Used primarily to differentiate Enterococcus Used primarily to differentiate Enterococcus

from Streptococcus

Members of Enterococcus are capable of

growing in the presence of 4% bile (oxgall)

and hydrolyzing esculin to glucose and

esculetin.

Bile Esculin Agar

Bile Esculin Agar (BEA)

Esculetin combines with ferric ions to produce

a black complex.

Certain bacteria are able to hydrolyze esculin.

A plate containing esculin will fluoresce a pale A plate containing esculin will fluoresce a pale

blue under UV radiation

Some bacteria can hydrolise this, leading to

UV dark colonies as opposed to UV light ones.

Thiosulphate Citrate Bile Salt Sucrose

(TCBS) Agar

Selective isolation medium for pathogenic

Vibrio sp.

Most Enterobacteriaceae other than Vibrio sp.

are suppressed for at least 24hrare suppressed for at least 24hr

Bile salts inhibit Gram-positive organisms

Sodium thiosulphate serves as a source of

sulphur, which in combination with ferric

citrate, detects hydrogen sulphide production

Thiosulphate Citrate Bile Salt

Sucrose (TCBS) Agar

When sucrose is fermented, it produces acid

which changes the pH

This is indicated by bromothymol blue and

thymol bluethymol blue

The medium is alkaline which enhances the

recovery of Vibrio cholerae

Saccharose (sucrose) is included as a

fermentable carbohydrate for the metabolism

of vibrios

Thiosulphate Citrate Bile Salt

Sucrose (TCBS) Agar

Cysteine Lactose Electrolyte Deficient

Medium (CLED) Agar

A valuable non-inhibitory growth medium

used in the isolation and differentiation of

urinary organisms

Being electrolyte deficient, it prevents the Being electrolyte deficient, it prevents the

swarming of Proteus sp.

Cysteine promotes the formation of cysteine-

dependent dwarf colonies

Cysteine Lactose Electrolyte Deficient

Medium (CLED) Agar

Lactose fermenters produce yellow colonies

on CLED agar; non-lactose fermenters appear

blue

It has a pH of approximately 7.3 It has a pH of approximately 7.3

Hektoen Enteric Agar (HEK / HE / HEA)

Selective and differential agar primarily used to

recover Salmonella and Shigella from patient

specimens

HEA contains indicators of lactose fermentation HEA contains indicators of lactose fermentation

and H2S production; as well as inhibitors to

prevent the growth of Gram positive bacteria

The pattern of lactose fermentation and H2S

production aids in the identification of the

organism subbed to the plate

Hektoen Enteric Agar (HEK / HE / HEA)

Luria-Bertani or Lysogeny Broth (LB)

LB is a nutritionally rich medium and is

primarily used for the growth of bacteria

Ingredients used to promote growth, include:

Peptides and casein peptones Peptides and casein peptones

Vitamins (including B vitamins)

Trace elements (e.g. nitrogen, sulfur, magnesium)

Minerals

Nutrient Agar / Broth

A microbiological growth medium commonly

used for the routine cultivation of non-

fastidious bacteria

It is useful because it remains solid even at It is useful because it remains solid even at

relatively high temperatures

Also, bacteria grown in nutrient agar grows on

the surface, and is clearly visible as small

colonies

Nutrient Agar / Broth

Ingredients:

0.5% peptone

0.3% beef extract

1.5% agar 1.5% agar

pH adjusted to neutral at

25C

Liquid media / broth are

used for growth of pure

culture

Nutrient Agar / Broth

In nutrient broth, the bacteria grows in the

liquid, and is seen as a soupy substance - NOT

as clearly distinguishable clumps

When bacteria are grown in broth, they may When bacteria are grown in broth, they may

exhibit patterns of growth ranging from a

sediment at the bottom of the tube, turbid

growth throughout the tube, or a pellicle

Nutrient Agar / Broth

Pellicle formation is sometimes due to:

affinity for oxygen

but may also be the result of hydrophobic

compounds present in the cell wall compounds present in the cell wall

or the general formation of dry, light colonies

If an organism produces and releases soluble

pigments, these will spread into the broth and

then change its colour

Slant Agar

Growth of bacterial culture on agar slants and

in broths can provide us with useful

information concerning:

motility motility

pigmentation

oxygen requirement

Slant Agar

Bacterial growth on slants depends on the

motility of the microorganism and ranges

from:

even (following the line of the original streak) even (following the line of the original streak)

irregular (slight spreading from the original line)

spreading (the organisms cover the entire surface

of the slant)

Slant Agar

Non-motile bacteria such as cocci usually

produce even slants when viewed after 24 - 48

hours of incubation

Slightly motile to motile species produce Slightly motile to motile species produce

irregular to spreading growth

Trypticase Soy Agar

The medium contains enzymatic digests of

casein and soybean meal

This provides amino acids and other

nitrogenous substances making it a nutritious nitrogenous substances making it a nutritious

medium for a variety of microorganisms

Dextrose is the energy source

Sodium chloride maintains the osmotic

equilibrium, while dipotassium phosphate acts

as buffer to maintain pH

Cetrimide Agar

Selective isolation of the Gram negative

bacteria Pseudomonas aeruginosa

Cetrimide is a selective agent against alternate

microbial floramicrobial flora

It also enhances the production of

Pseudomonas pigments such as pyocannin and

fluorescein, which show a characteristic blue-

green and yellow-green colour, respectively