Lecture 2

Question 1

three properties of media

Answer 1

Physical state:medium’s normal consistency
Chemical comp.:type of chemical medium contains
Functional : purpose of the medium

Question 2

LIQUID MEDIA

Answer 2

They are defined as water-based solutions, that do not solidify at temperature above freezing and flow freely when the container is tilted
They are termed as broths or infusions
A common lab medium is nutrient broth/TSB
Growth occurs throughout the container in the form of dispersed, cloud or particulate appearance.
Growth in broth means – turbid

Question 3

Semi-solid:

Answer 3

At Rt semi-solid media show a clot like consistency
This is due to the presence of a solidifying agents like agar or gelatin, that thickens the medium
Semi-solid medium is used to determine the motility of bacteria or to localize a reaction at a specific site e.g. SIM medium

Question 4

Solid medium

Answer 4

They provide a firm surface on which bacterial cells can grow.
Cells growing on a solid medium are termed as a colony and are visible to naked eye.
One colony is formed from a single cell (growth) and so consists of just one species and no other
Use colony only on petri plates
e.g of solid medium:
-Slant in a test tube
-Stab or deep in a test tube
-plate in a Petri dish

Question 5

Slants used to?

Answer 5

used to maintain stock cultures

Question 6

Deep is used to ?

Answer 6

observe motility and for anaerobic growth

Question 7

Colony characteristics used for the identification purpose.

Answer 7

Size,shape,color,like,moistness or dry

elevation-raised or flat

Question 8

AGAR

Answer 8

It is a complex polysaccharide isolated from red algae .
it is solid @ Rt and melts or liquefies at boiling point of water( 100 oC)
Once liquefied it does not resolidify, until it cools to 42 oC
So if a liquefiable form is required, the sterile medium is liquefied(boiled at 100 oC) and kept in a water bath @ 50-56o C .This is known as tempering the medium
Normal concentration of agar is between 1 to 5% ,
1.2 to 1.5 % being the most common, depending upon the media requirement
Gelatin if used as a solidifying agent ,has to have a concentration of 10 % to 15 %
but then also will not remain solid as satisfactory as agar.

Question 9

Synthetic Media

Answer 9

kind and quantity of each ingredient is known

Used mainly for tissue culture media.

Question 10

Non-Synthetic Media:

Answer 10

do not know the exact kind and quantity of each ingredient
Has animal or human derived components
Eg: yeast extract,peptones ,calf heart meat,sheep blood,bovine serum etc.

Question 11

General purpose medium

Answer 11

They are designed to grow as broad a spectrum of microbes as possible.
As a general rule they are non synthetic and contain a mixture of nutrients that could support a variety of microbes.
E.g Nutrient broth,N.agar,TSA-trypticase soy agar

Question 12

Enriched medium

Answer 12

non selective
It is pre prepared media to which additional growth factors have been added.
e.g Blood agar .It is one of the most frequently used media in clinical bacteriology
It is used to recover a number of organism which are unable to grow on basic medium and also to study the hemolysis reactions
( α,β or no reaction )

Question 13

Blood agar

Answer 13

is prepared by adding sterile 5% sheep blood to TSA base

Since used to detect bacteria that produce hemolysin ,It is also a differential medium

Question 14

Alpha hemolyisis-

Answer 14

On blood agar
It is a type of hemolysis which is characterized by a zone of greenish clearing, due to the incomplete breakdown of the hemoglobin
Species of Streptococcus that are part of the normal flora of the throat are all alpha hemolytic, Streptococcus pneumonia
alpha hemolytic

Question 15

Beta hemolysis

Answer 15

On blood agar
It is characterised by a clear zone around the colony due to hemolysis ie lysis of the blood cells
E.g Streptococcus pyogenes , is beta hemolytic
this org.causes strep. Throat

Question 16

Gamma hemolysis

Answer 16

– no lyse at all

Question 17

lysed blood

Answer 17

Another form of blood used for enrichment
Lysis releases cellular constituents making more nutritious elements available to bacteria for growth
e.g chocolate agar

Question 18

Chocolate agar

Answer 18

it is prepared by heating the blood at 85oC .It has 10% of blood.
The increased heat lyses the RBCs causing the medium to take a brown or chocolate color.
More enrichment medium than differential
Lysis releases,certain factors from the RBC into the medium like Hemin (factor X) NAD (factor V):Nicotine Amide Dinucleotide Org.like
Haemophilus influenzae – sore throat in kids
Neisseria – STI in vagina require these factors to grow.

Question 19

Thayer-Martin

Answer 19

agar is chocolate agar made selective by the addition of antibiotics.
It is used for primarily isolating Neisseria bacteria namely N. gonorrhoeae and N. meningitidis from specimens containing mixed flora taken from the throat, vagina,rectum and urethra.
Consisting of Chocolate Agar with antibiotics such as vancomycin, colistin and nystatin,

Question 20

Vancomycin

Answer 20

is able to kill all gram –positive organisms

Question 21

Colistin

Answer 21

able to kill Gram-negative organism except Neisseria and not very active against Proteus

Question 22

Nystatin

Answer 22

able to kill all fungi

Question 23

Modified Thayer-Martin (MTM II) Agar also contains

Answer 23

trimethoprim for the inhibition of Proteus spp

Question 24

Selective Medium

Answer 24

It is a medium that may have certain agents that allows the growth of certain bacteria and Inhibit certain bacteria.

Question 25

Differential Medium

Answer 25

It is a medium that may have certain agents that allows the differentiation of different bacteria that can grow on the medium
Eg. MacConkey agar.MAC – mostly on urine and poop, gram negative
lactose positive sample turns purple on agar

This medium is used to isolate and differentiate member of the Enterobacteriaceae (all gram negative and rod shaped, found in gut), based on the ability to ferment lactose.
Most of the differential media are also selective media

Question 26

selective agents

Answer 26

Bile salts, oxgall and crystal violet They inhibit growth of gram-positive organisms

In this medium bile salts and brilliant green dye are the selective agents against gram positive and many other gram negative organisms

Question 27

Lactose is

Answer 27

differentiating agent in MAC, there are certain agent cannot ferment lactose where others can. For example Ecoli can ferment it
When sugar is fermented acid and gas are produced.
There are pH indicators such as phenol red is added to MAC to see when there is acid production causing the colonies to have a pink color. Non fermenters give beige colonies

Question 28

MacConkey Medium

Answer 28

Differentiation
Lactose is a fermentable carbohydrate.
When lactose is fermented, acid is produced which causes a local pH drop around the colony that causes a color change in the pH indicator (neutral red) and bile precipitation.

Report as Lactose Fermenters or
Non Lactose Fermenters
LF is pink/red
NLF is clear or yellowish

Question 29

Salmonell-Shigella medium

Answer 29

This is another example of differential/ selective medium
Still Enterobacteriaceae (therefore gram negative and rod shaped)
It is a selective medium used for the isolation of Salmonella and many Shigella species,i.e nonlactose fermenting enterics from lactose fermenting enterics.
Contains Lactose and Na thiosulfate as the differentiating agents
Neutral red as pH indicator
Agar as solidifying agent
OH:157H7 – on uncooked meat found on rectal swabs
Salmonella through sewage and poop

Question 30

Describe diff agents in SS medium

Answer 30

Lactose (utilizable sugar)
Organism like E.coli that ferment lactose produce pink colonies,NLFs produce colorless colonies
Sodium thiosulphate and ferric citrate ( source of reducible sulphur),enable the detection of Hydrogen sulphide by black colonies.
Na thiosulphate and Ferric citrate are a much stronger differential agent.
Salmonella can reduce sulphur ,produce H2S and give black colonies
Shigella can not reduce sulphur,hence is H2S negative and do not give black colonies

Salmonella can reduce NA thio producing Hydrogen sulfate giving black colored colonies in the presence of ferric citrate
Shigella cannot reduce NA thio and does not give off black colored colonies by producing hydrogen sulfate

Question 31

Hektoen Enteric (HE) Agar

Answer 31

better for shigella
Is a moderately selective medium used in the isolation of gram-negative enteric microorganisms, especially
Salmonella and Shigella
The selective nature of Hektoen Enteric Agar is due to the addition of bile salts.
These inhibits gram-positive organisms but also can be toxic for some gram-negative strains

Question 32

HEK MEDIUM

Answer 32

This medium contains three carbohydrates,
lactose, sucrose and salicin.
Lactose concentration is higher.Lactose fermenters give salmon colored colonies
The indicator system, consisting of
Acid fuchsin and bromthymol blue
Ferric ammonium citrate and sodium thiosulphate are the differential agents enable the detection of
hydrogen sulfide production the differentiation process due to the production of black- centered colonies. Salmonella gives black colonies ,Shigella does not

best for salmonella

Question 33

PRAS:

Answer 33

pre-reduced and anaerobically sterilized medium.
Reducing agent in such medium is L-cysteine hydrochloride.
PRAS is made in the absence of oxygen using special equipment.

Question 34

PEA-PhenylEthyl Alcohol Agar

Answer 34

Phenylethyl Alcohol is inhibitory to gram negative
selective medium for Gram positive organism
org.like E.coli,since it selectively and reversibly inhibits DNA synthesis.
Gram positives like S.aureus grow well due to the presence of peptones which supply nitrogen,carbon,sulphur and trace elements

Question 35

Chromogenic Medium (screening media):

Answer 35

This is the latest in the development of the culture media
At the moment mainly used for UTI pathogens
The use of chromogenic substrates in the culture media has shown to increase the microbial identification directly from the medium.
Chromogenic substrates have proved to be a powerful tool in the identification of microorganisms
due to the detection of specific enzymes produced by the target microorganism.
These enzymes break the chromogenic substrate that points up the microorganism by color differentiation of the grown bacteria
Differential medium tells you what it can be – lactose fermentor or not, whereas on Chromogenic narrows down to what is growing – this is Ecoli , this is MRSA

Question 36

α-glucosidase

Answer 36

produced by Staphylococcus aureus cleaves the chromogenic substrate and gives a blue color to the Staphylococcus aureus colony.
MRSA agar - Chromogenic medium -

Question 37

Cefoxitin

Answer 37

Supplement inhibits the growth of Staphylococcus aureus sensitive to methicilin.
Results in 24 h.
Easy Interpretation by color of colonies:
in Chromogenic medium -

Question 38

Primary Plating Medium(PPM)

Answer 38

This is a medium first inoculated with the specimen collected from the patient.
The primary medium/media is/are chosen based on the site of infection and population of the suspected organisms.
PPM for wound can be different from that of a urine or a throat swab specimen
PPM can be just one medium or 3-4 different media

Question 39

Sorbitol MacConkey

Answer 39

Sugar is Sorbitol
Report as Sorbitol Fermenters or
Non Sorbitol Fermenters
SF is pink/red -NSF is clear or yellowish

Question 40

Hektoen

Answer 40

2 sugars: SUCROSE & SALICIN
Fermenters appear yellow or orange
Non-Fermenters appear Clear green or blue
Bile salts are the selctive agents which inhibit the gram positive organisms.
For both, you may see blackening which is H2S

Question 41

Salmonella-Shigella (SS)

Answer 41

Sugar is also lactose
Fermenters appear as pink
Non fermenters appear as clear/pale yellow
May also detect H2S by sodium thiosulfate which appears as black centers in the colony
Bile salts and brilliant green dye are the selective agent
Sodium thiofulfate is the differential agent