Week 2: Culturing Microorganisms

Question 1

Aseptic technique

Answer 1

process and procedures that reduce the chance of contamination.

Question 2

Colony forming unit

Answer 2

Singular cell or small group of related cells possible of producing a colony.

Question 3

Clone

Answer 3

the derived cells from the original colony forming units.

Question 4

Culture

Answer 4

he propagation of microorganisms in a growth medium

Question 5

Fastidious

Answer 5

any organism that has complex or particular nutritional requirements

Question 6

Incubation

Answer 6

favorable conditions that promote microorganism to grow.

Question 7

Inoculum

Answer 7

original sample being used, the sample you’re taking from.

Question 8

Medium/media

Answer 8

Collection of nutrients that fosters the growth of a sample.

Question 9

Normal microbiota

Answer 9

microorganisms associated with a particular area of the body, normally not disease causing.

Question 10

Pure culture

Answer 10

a population of cells or multicellular organisms growing in the absence of other species or types.

Question 11

Sterile

Answer 11

competing completely free of microorganisms.

Question 12

Define and contrast “colony forming unit and clones”

Answer 12

Colony forming unit is the singular or small group of starting cells that grow a multiple to form a colony consisting of clones.

Question 13

Define and contrast “inocoulum and inoculation”

Answer 13

Inoculum is the original sample being used and inoculation is the process of transferring this sample for further growth.

Question 14

Define and contrast “mixed and pure culture”

Answer 14

Mixed culture: how microorganisms occur in nature, difficult to study.

Pure culture: Isolated microorganisms, necessary for study and characterization, important for diagnosis of infectious disease, and necessary to satisfy Koch’s Postulates.

Question 15

Do microorganisms normally grow in pure culture in nature? Why or why not?

Answer 15

No, Normally in mixed cultures. Multiple species grow in the same environment and many work together for different processes.

Question 16

Why is it important to isolate and cultivate microorganisms in pure culture?

Answer 16

Easier to study and characterize, eliminating the variable of other microorganisms when looking at processes and conducting experiments.

Question 17

What is aseptic technique?

Answer 17

Series of processes and procedures used to minimize contamination.

Question 18

Aseptic technique is sometimes called sterile technique. Why is the latter term inappropriate?

Answer 18

Sterile means the absence of microorganisms, the aseptic technique is not a sterile process overall since you are picking up specific microorganisms and moving them. There are times when sterilization occurs so that you can intentionally pick up your desired organisms without contamination but the process overall is not sterile.

Question 19

List at least three different clinical specimens, including the methods of collection.

Answer 19

Blood, is drawn into sterile tubes that contain different clotting or anticoagulant mediums.

Urine, can be a sterile sample taken via cysto or non-sterile via free catch. Placed into a sterile container or test tube.

Cerebrospinal fluid, taken via a sterile spinal tap procedure.

Question 20

What two important qualities are does transport media possess?

Answer 20

Maintains appropriate environment for specimen survival and maintains relative abundance of different microbes.

Question 21

What is the purpose of a streak plate?

Answer 21

to obtain isolated colonies from an inoculum by creating areas of increasing dilution on a single plate.

Question 22

Describe the process of making a streak plate, including steps related to aseptic technique. Where on the plate are you most likely to find isolated colonies?

Answer 22

Sterilize loop with flame, sterilize neck of test tube with flame and open without placing cap down. Sterilize the neck of the tube again and replace cap.Pickup inoculum and create streaking pattern 1. Sterilize the loop again and starting from sterak 1 makes streaking section 2. Repeat for another 1-2 more streaks starting from the previous streak made. Close petri dish and incubate. Sterilize loop before putting away.

Most likely to find isolated colonies in area 4.

Question 23

What is the purpose of a pour plate? How is it like a streak plate and how does it differ from a streak plate?

Answer 23

Purpose is to grow isolated colonies of microbes through a series of dilutions. Like a streak plate in that you are using a process to dilute the sample. Different in that pour plates you are diluting in a liquid agar media before pouring into plate for solidification. Also different in that you will end up with multiple plate instead of just using one.

Question 24

What is a serial dilution (series of dilutions)?

Answer 24

a stepwise series of dilutions that are performed to reduce the concentration of a substance in a solution to a more usable concentration

Question 25

Describe the process of making a pour plate, including steps related to aseptic technique. Are there any particular locations in or on the plate where you are most likely to find isolated colonies? Are there any particular locations in or on the plate where colonies might be easier to retrieve?

Answer 25

Take from initial sample and mi into liquid media containing agar, sterilize loop then take for the first tube and imbed into the second dube. Pure solution into dis and allowed to solidify and incubate. Most cells are embedded within the solid media but colonies at the top are easier to retrieve.

Question 26

What types of organisms can be effectively isolated using streak and/or pour plates? What types of organisms are not effectively isolated in these ways, and why? Describe how the latter groups of organisms can be effectively isolated.

Answer 26

Streak and pour plates are useful for bacteria and fungi. Protozoa and motile unicellular algae does not work well since they do not stay in one location when forming colonies. They work better in broth media. Larger microbes can be isolated with pipette while under slight magnification.

Question 27

What are the basic requirements for culture media? Why do you think we are unable to grow most microorganisms in culture media?

Answer 27

Walter, essential nutrients (organic and inorganic, energy source, ions/salts), appropriate oxygen level, other physical conditions (pH, osmotic pressure, temp).

Question 28

How does one convert liquid media into solid media?

Answer 28

Addition of Agar

Question 29

Solid media can be placed in various containers in various ways. Describe three such examples.

Answer 29

Slants: test tubes at an angle, creates gradient of thickness.

Deeps: test tubes full of solid media.

Petri plates: thin layer of solid medium but increased surface area.

Question 30

Why should the caps on culture tubes remain loose?

Answer 30

To allow gas exchange.

Question 31

What is agar? From where is it obtained? For what basic reason do we add agar to liquid media?

Answer 31

Complex polysaccharide derived from cell walls of certain red algae. Added to liquid media so it dissolves.

Question 32

What features of agar make it very useful to microbiology?

Answer 32

Resistant to microbial degradation; does not act as nutrient source and microbes cannot digest it. Translucent making colonies easy to see.

Question 33

At approximately what temperature does agar melt? At approximately what temperature does agar solidify?

Answer 33

Melts near boiling, solidifies below 40 C.

Question 34

How are complex and defined media similar? How are they different?

Answer 34

complex: “non-synthetic media”, contains a variety of ingredients at unknown concentrations, great for routine purposes.

Defined: “synthetic medium”, compost of precise amounts of pure chemicals, used for specific purposes.

Difference is weather concentrations are known vs unknown.

Question 35

What is peptone? How is it made?

Answer 35

Complex media, mixture of short peptides and amino acids. Produced by digesting protein from a variety of sources by various means.

Question 36

Describe two different means by which obligate anaerobes can be successfully cultured.

Answer 36

Oxygen free depths of solid media “slab culture”.

Reducing media, thioglycollate chemical compound added that chemically combines with O2 removing it. Heat used to drive off oxygen immediately before inoculation.

Petri plate inside sealable containers containing reducing chemicals.

Question 37

How difficult is it to grow T. pallidum and M. leprae on culture media? How do we grow them?

Answer 37

Impossible to grow on a culture medium so animal or cell cultures are used. T.pallidum “syphilis” in rabbits and M.leprae “ leprosy” in armadillos.

Question 38

Why is it more challenging to grow organisms such as N. gonorrhoeae and H. influenzae on culture media than it is to grow E. coli?

Answer 38

They have specific nutrient requirements including specific growth factors.

Question 39

How difficult is it to grow E. coli on culture media? Why?

Answer 39

Very easy, not particular regarding nutrient needs.

Question 40

What is the purpose of selective media? What are some ways in which this is accomplished?

Answer 40

Selective media favors growth of specific bacteria while suppressing the growth of other bacteria. Accomplished by removing specific nutrients or adding specific nutrients.

Question 41

What is the purpose of differential media? What are some ways in which this is accomplished?

Answer 41

Differential media causes a visible appearance change in the presence of specific bacteria, either change in the colonies appearance or media appearance. Accomplished by adding indicators like pH indicators of blood agar.

Question 42

Discuss at least three specific examples of selective media, explaining the basis of the selectivity.

Answer 42

High Nacl concentrations- bacteria has to have to be salt tolerant to grow.
Acidic pH- inhibits bacterial growth and makes media selective for fungi.
Media lacking nitrogen- only nitrogen-fixing bacteria can grow.

Question 43

Discuss at least three specific examples of differential media, explaining the basis of the differentiation.

Answer 43

pH indicator like phenol red- turns yellow under acidic
conditions, indicates acid formation.

Blood agar- detect bacteria that produces hemolysin which lyses red blood cells.

MacConkey Agar- lactose fermentation causes color change. Lactose fermenting bacteria colonies are red or pink and turn the media red.

Question 44

Give at least two examples of media that is both selective and differential, explaining the basis for each.

Answer 44

Mannitol salt agar- selective for salt tolerant microorganisms as it contains high NaCl concentration. Differential by containing sugar mannitol and pH indicator phenol red. Fermentation produces acid and turns media yellow. No fermentation, no color change.

Eosin methylene blue- selective for gram-negative bacteria, inhibits gram-positive bacteria. Differential for lactose fermentation which decrease pH of the media. Slow lactose fermenters produce dark pink color and fast lactose fermenters produce dark purple color (with green metallic sheen).

Question 45

Why is some media enriched with blood?

Answer 45

to provide additional growth factors for specific bacteria and can also be used to make differential media.

Question 46

What is beef extract? What is yeast extract? How is each made? Why do you think these extracts are particularly helpful in culturing microorganisms?

Answer 46

Complex media, water soluble components of a substance. Provide minerals, vitamins, and other nutrients for microorganisms.

Question 47

What microbe is commonly cultured in rabbits?

Answer 47

Treponema pallidum, syphills

Question 48

What microbe is commonly cultured in armadillos?

Answer 48

Mycobacterium leprae, leprosy