Culture Media

Question 1

Why is agar considered a solidifying agent?

Answer 1

1. It isn’t degraded by bacteria
2. It consists of no useful nutrients for bacteria
3. Liquid state at high temperatures and solid state at temperatures ideal for bacterial growth

Question 2

Broths use

Answer 2

• Identifying growth patterns in liquid media, biochemical tests, inoculations and cultivating large amounts of bacteria

Question 3

Agar slant use

Answer 3

• Generating bacterial stocks which are used for freezing bacteria

Question 4

Agar plates use

Answer 4

• Observation of colony features for various bacterial species

Question 5

Growth media can be both selective and differential, give an example

Answer 5

EMB (eosin methylene blue agar) inhibits the growth of Gram positive bacteria. Gram negative bacteria that grow on this medium are differentiated based on their ability to ferment the sugars lactose and sucrose.

Question 6

Aseptic technique of inoculating

Answer 6

If you are inoculating a tube of broth or an agar
slant,
- remove the cap of the tube (do not set the cap down on the table) and flame the lip of the tube.
- Throughout the procedure, hold the tube at an angle to reduce the probability of particles entering the opening.
- Insert the loop into the tube and transfer bacteria to the growth medium.
- Be careful that only the sterilized part
of the loop touches the tube or enters the growth medium
- Flame the lip of the test tube before replacing the cap.

Question 7

Pure culture

Answer 7

• contains single bacterial species

Question 8

Mixed culture

Answer 8

• contains various types of bacterial species

Question 9

Why place plates inverted for incubation?

Answer 9

Condensation occurs when the warm air rises, then cools and as such loses the ability to hold on to water vapor.
Plates are incubated for prevention of condensation, which causes water to fall on agar surface and disrupt streaking patterns.

Question 10

Tryptic soy agar (TSA):

Answer 10

General purpose complex growth medium.

Question 11

Mannitol-salt agar (MSA):

Answer 11

Differential and selective growth medium.

Question 12

MSA - purpose

Answer 12

It is selective for staphylococci due to the high concentration of NaCl, and differentiates based on the ability to ferment mannitol.
Staphylococci that ferment mannitol produce acidic byproducts that cause the phenol red to turn yellow.
This produces a yellow halo in the medium around
the bacterial growth.

Question 13

MSA interpretation - selective aspect

Answer 13

Selectivity
Growth - interpretation (organism not inhibited by NaCl) - ID Staphylococci, Micrococcus
No growth - interpretation (organism is inhibited by NaCl - ID Non Staphylococcus spp

Question 14

MSA interpretation - differential aspect

Answer 14

Yellow halo - Interpretation (bacteria ferments mannitol) - ID S aureus
Yellow halo absent - Interpretation (bacteria does not ferment mannitol) - ID Staphylococcus spp (not S aureus), Micrococcus (yellow colonies)

Question 15

EMB - Selective and differential – describe selective aspect

Answer 15

Methylene blue and Eosin Y are contained in this medium. The dyes inhibit gram positive bacteria. Medium is thus selective for the gram negative bacteria.

Question 16

EMB - Selective and differential – describe differential aspect

Answer 16

Medium differentiates on sucrose and/or lactose fermentation.
If organism does not ferment either sugar, colourless colonies are formed.

Question 17

The levels of fermentation differ depending on type of bacteria. Describe effects of fermentation on colonies.

Answer 17

Fecal coliforms (intestinal bacteria) generate large quantities of acidfrom the fermentation of sucrose or and/or lactose.
Organisms producing some acid will display as purple or pink colonies
Organisms producing much more acid will display purple to black colonies in a green metallic sheen.

Question 18

Coliform vs fecal coliform

Answer 18

Coliformbacteria live in the soil, and these organisms may be the source of those that appear in the water, especially surface water.
Fecal coliforms, on the other hand, are more specific because they refer to thecoliformsthat live in the intestinal tract of humans and many other animals.

Question 19

Interpretation EMB

Answer 19

Pink and mucoid colonies
Dark purple to black with/without green metallic sheen
Growth pink and mucoid - organism ferments sucrose and/or lactose generating some acid production. - coliform bacteria
Growth dark purple to black with/without green metallic sheen - organism ferments sucrose and/or lactose with large acid production - Fecal coliform e.g. E. coli

Question 20

What general type of growth medium would you use to:

(a) grow one type of bacteria but inhibit the growth of another type?

Answer 20

Selective media is used to facilitate growth of certain organisms whilst inhibiting growth of others.

Question 21

What general type of growth medium would you use to:

(b) discriminate between different types of bacteria?

Answer 21

Differential media are utilized for differentiating organisms that are closely related or belong to the same group.

Question 22

A bacterial species is inoculated on EMB agar.

(a) The bacteria do not grow. Why?
(b) If the bacteria ferment lactose, what would you expect to see?
(c) The bacteria produce clear colonies. Why?

Answer 22

Because growth may be inhibited by the dyes contained in EMB agar, organism may be gram positive.

Pink and mucoid colonies or purple to black colonies with metallic green sheer
Because bacteria does not ferment lactose

Question 23

If you were testing water for the presence of fecal coliforms, what sort of medium would you use: TSA,
EMB agar or MS agar? If fecal coliforms were present, what would their growth characteristics be on this
medium?

Answer 23

EMB
Fecal coliforms ferment sucrose and/or lactose and generate a high amount of acid byproducts. This causes the medium to change to purple to black with/without green metallic sheen.

Question 24

Examples of Selective media

Answer 24

Eosin methylene blue contains dyes that are toxic for Gram-positive bacteria.

YM (yeast extract, malt extract agar) has a low pH, deterring bacterial growth.

MacConkey agar is for Gram-negative bacteria.
Hektoen enteric agar is selective for Gram-negative bacteria.

Mannitol salt agaris selective for gram-positive bacteria and differential for mannitol.

Xylose lysine deoxycholateis selective for Gram-negative bacteria.

Buffered charcoal yeast extract agaris selective for certain gram-negative bacteria, especiallyLegionella pneumophila.

Sabouraud’s agaris selective to certain fungi due to its low pH (5.6) and high glucose concentration (3–4%).

Question 25

Examples of differential media

Answer 25

Blood agar(used instreptests) contains bovine heart blood that becomes transparent in the presence of β-hemolytic organisms such asStreptococcus pyogenesandStaphylococcus aureus.

Eosin methylene blueis differential for lactose fermentation.

Granada mediumis selective and differential forStreptococcus agalactiae(group B streptococcus) which grows as distinctive red colonies in this medium.

MacConkey agaris differential for lactose fermentation.

Mannitol salt agaris differential for mannitol fermentation.

Question 26

Why is it necessary to sterilize the loop between streaks when streaking for single colonies?

Answer 26

• Streak plate method involves diluting the bacteria to low enough amounts that individual colonies can be acquired.

A colony is essentially a bacterial clump growing from a single cell. In order to obtain this, we must spatially arrange the bacteria with enough space that the colonies are not overlapping.
Without flaming between each streak, there will be a constant addition of bacteria onto the loop for every streak.

Also, if the loop is not flamed then there is a higher chance of contamination of culture. The more the loop is waved around the higher the chance of bacteria landing and infiltrating plates.