Week 1, Intro to microbes and their growth requirements

Question 1

List some types of microorganisms

Answer 1

> Bacteria > Fungi > Protozoa > Algae > Viruses > Viroids > Prions

Question 2

Provide the classification, based on atmospheric (oxygen) requirements), the organism in test tube (a)

Answer 2

Obligate aerobe

Question 3

What units are commonly used to measure microbes?

Answer 3

> Micrometer (µm) = 0.001 mm = 10⁻⁶ m > Nanometer (nm) = 0.001 µm = 10⁻⁶ mm = 10⁻⁹ m

Question 4

How do most fungi reproduce?

Answer 4

Spores, hyphae.

Question 5

Explain how microbes are grouped by their energy, electron, and carbon sources.

Answer 5

ENERGY SOURCE - Light (Phototrophs) - Organic or inorganic compounds (by oxidation) (Chemotrophs) ELECTRON SOURCE - Organic molecules (Organotrophs) - Reduced inorganic molecules (Lithotrophs) CARBON SOURCE - CO2 (Autotrophs) - Organic molecules (reduced, preformed, from other organisms, etc) (Heterotrophs)

Question 6

What does the term ‘resolution’ refer to?

Answer 6

The ability to distinguish two objects as distinct and separate Human eye: 100-200 µm Light microscope: ~200 nm Electron microscope: ~0.5 nm

Question 7

Explain the method for sub-culturing filamentous fungi

Answer 7

1. Label the under-side rim of a fresh potato dextrose agar (PDA) plate 2. Dip the inoculating needle or scalpel into alcohol, flame, cool. 3. Cut a small block (approx 5mm x 5mm) from the actively-growing edge of the mycellium 4. Transfer to the center of the PDA plate 5. Incubate right way up for 1 week at 25 °C

Question 8

Provide the classification, based on atmospheric (oxygen) requirements), the organism in test tube (b)

Answer 8

Obligate anaerobe

Question 9

What are some of the roles of microorganisms?

Answer 9

> Nutrient cycling > Waste treatment > Disease and biological control agents > Food and beverage industry (fermentation) > Pharmaceutical industry (antibiotics) > Oil and petrochemical industry (ethanol) > Biotechnology > Understanding basic biology

Question 10

T/F: Sterilisation degrades vitamins

Answer 10

True

Question 11

Desribe what a Photolithoautotroph is and give an example of one.

Answer 11

Cyanobacteria is an organism that uses light as its energy source, reduced inorganic molecules for its electron source, and CO2 as its carbon source.

Question 12

When using asceptic technique, the goal is to kill the most resistant microbial component, which is…?

Answer 12

Bacterial endospores.

Question 13

What are four sterilisation techniques?

Answer 13

HEAT - Dry heat → denatures microbial proteins → metal utensils: flamed to red-hot → glassware: heat to 160-170 °C in oven → less effective than moist heat - Moist heat → e.g. autoclave (steam under pressure) → 121 °C at 15 psi for 15-20 minutes → glassware, aqeous solutions, disposal of unwanted cultures CHEMICALS - Gases → e.g. ethylene oxide (penetrating, kills all microbes) - Alcohols, e.g. ethanol - Phenolics, e.g. triclosan (hand sanitisers) - Halogens, e.g. bleach, iodine - Heavy metals, e.g. silver - Essential oils, e.g. tea tree oil RADIATION - Ionising radiation → gamma rays, electron beams → lethal, good penetration - UV light → surfaces only (doesn’t penetrate surfaces) * Approved by the FDA and WHO for use on meat, fresh fruit and vegetables, and spices. FILTRATION - for heat-labile liquids (damaged by heat) - for air (HEPA filter, laminar flow hood)

Question 14

List 3 defining features of microorganisms

Answer 14

1. < 1 mm diameter 2. They have a common method of study (microscopy) across different groups of microbes 3. Simple organisation (single cells or cell clusters, or sub-cellular)

Question 15

Provide the classification, based on atmospheric (oxygen) requirements), the organism in test tube (e)

Answer 15

Aerotolerant anaerobes

(anaerobic regardless of [O₂], e.g. LAB)

Question 16

T/F: Sterilisation degrades antibiotics

Answer 16

True (denatures)

Question 17

What is meant by a ‘selective medium’?

Answer 17

A culture medium designed to promote (and/or inhibit) certain species of microbes; e.g. using antibacterial antibiotics to select for fungi.

Question 18

List some factors that affect microbial growth

Answer 18

• Temperature - pH - Osmotic pressure - Atmosphere

Question 19

T/F: Sterilisation degrades degrade agar.

Answer 19

False

Question 20

Explain the (streak plate) method for establishing pure bacterial cultures.

Answer 20

1. Label the under-side of the petri dishes and mark the location of the first streak 2. Flame and cool a loop 3. Collect a loop-full of bacterial culture 4. Gently wipe the loop over one side of the agar 5. Flame and cool loop 6. Turn dish anti-clockwise and make 4-5 gentle parallel strokes in one direction through the previous set of strokes 7. Flame and cool loop 8. Repeat steps 6 & 7 twice (or once, then stroke in a zig-zag pattern towards the center) 9. Incubate inverted for 1 week at 25 °C 10. Select an individual colony from the incubated culture and streak on a new agar plate. Repeat as many times as necessary.

Question 21

What temperature does agar melt and solidify at?

Answer 21

Melts at 100 °C, sets at 42 - 44 °C.

Question 22

Describe a typical growth curve for single-celled microbes.

Answer 22

Log # of viable cells OVER time 1. LAG PHASE: adjusting to new conditions, synthesising new materials; variable duration 2. LOG (EXPONENTIAL) PHASE: number of cells doubles at a constant rate (1 -> 2 -> 2ⁿ); after n generations, population, N=N₀2ⁿ; population most uniform (chemical and physiological). 3. STATIONARY PHASE: nutrient limitation, accumulation of toxic waste, 2° metabolites (could be lactic acid which changes the pH o the point that it becomes unfavourable, antibiotics produced) 4. DEATH PHASE:

Question 23

Explain what the term ‘viability’ refers to.

Answer 23

The ability of bacteria and viruses to reproduce, and the ability of filamentous fungi and algae to grow

Question 24

Explain the process for preparing agar plates for culture.

Answer 24

1. Spray and wipe down the work surface with bench cleaner 2. Label the petri dishes on their under-side, around the rim, and place them on the bench with the lids on top and writing underneath (i.e. the right way up) 3. Working quickly, dry the vessel containing the molten agar, remove the cap, flame the opening, raise the lid of the petri dish slightly, and pour in the agar. 4. Rotate the dish gently to evenly distribute the agar. 5. Leave the agar to set.

Question 25

Define sterilisation

Answer 25

Free from all living organisms

Question 26

Describe the difference between an obligate aerobe, a facultative aerobe, and a microaerophile.

Answer 26

OBLIGATE aerobes REQUIRE oxygen to survive. FACULTATIVE aerobes prefer (and grow better) in oxygen-rich environments, but can survive in low-oxygen environments. MICROAEROPHILES typically grow in environments with between 2-10% O2.

Question 27

What is the optimum pH for culturing bacteria?

Answer 27

pH 6-8 Nutrient agar is pH 6.8

Question 28

What is the optimum pH for culturing fungi?

Answer 28

~ pH 5 PDA is pH 5.6 Acidified PDA is 4.8; used for inhibiting bacteria

Question 29

T/F: Nematodes aren’t considered microbes.

Answer 29

True. All to do with organisation - nematodes have more complicated digestion, for example)

Question 30

Define microbiology

Answer 30

The study of organisms too small to be seen by the naked eye

Question 31

What is meant by a ‘differential medium’?

Answer 31

A culture medium designed to allow for easy identification of certain microbes based on their appearance; e.g. by containing dyes that cause the organism of interest to appear a particular colour.

Question 32

Explain what ‘death’ means in terms of microbes

Answer 32

The irreversible of the ability to reproduce or grow

Question 33

Explain the method for sub-culturing bacteria.

Answer 33

Basically just transfer bacterial culture from one tube/dish to another using aseptic technique.

Question 34

Define ‘sterile’.

Answer 34

Free from all living organisms

Question 35

How does studying microbes help with our understanding of basic biology?

Answer 35

> Easy to work with > Don’t require ethics approval > Microbes make up a large proportion of our own systems

Question 36

Explain the method for calibrating an ocular micrometer.

Answer 36

1. Place ocular micrometer in one of the eyepieces. 2. Place a stage micrometer on the microscope stage 3. Using the 10x objective, move the stage so that the ocular and stage scales are superimposed at the first increment. 4. Count the number of increments until the lines superimpose over each other again. 5. 1 ocular micrometer = (# ocular micrometer increments) / (# stage micrometer increments) * the distance between the smallest increments on the stage micrometer (µm) e.g: At 10x, 1 ocular unit = 99/100*10 = 9.9 µm At 40x, 1 ocular unit = 15/60*10 = 2.5 µm

Question 37

Provide the classification, based on atmospheric (oxygen) requirements), the organism in test tube (d)

Answer 37

Microaerophile

Question 38

Provide the classification, based on atmospheric (oxygen) requirements), the organism in test tube (c)

Answer 38

Facultative aerobe/anaerobe

Question 39

What is the main difference between nutrient agar (NA) and potato dextrose agar (PDA)?

Answer 39

pH. → NA: 6.8 → PDA: 5.6

Question 40

How are microbes measured?

Answer 40

> Total cell count > Viable count > Cell mass (turbity, cell volume, dry weight, chemical measurements)

Question 41

Describe what a Chemolithoautotroph is and give an example of one.

Answer 41

An organism that oxidises organic or inorganic compounds for its energy source, reduced inorganic molecules for its electron source, and CO2 for its carbon source; e.g. Nitrifying bacteria; methanogens; and S, H, and FE oxidisers

Question 42

How do most bacteria reproduce?

Answer 42

Binary fission or budding

Question 43

Explain what a ‘culture’ or microbes refers to.

Answer 43

A population of microbes grown outside of their natural environment (e.g. in a lab).

Question 44

Describe what a Chemoorganoheterotroph is and give an example of one.

Answer 44

An organism that oxidises organic or inorganic compounds for its energy source, organic molecules for their electron source, and organic molecules produced by other organisms for its carbon source; which includes most fungi, pathogens, protists, and archaea