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Biological Sciences > Workshop Lab Semester 1 Weeks 1-4 > Flashcards

Workshop Lab Semester 1 Weeks 1-4 Flashcards

1
Q

All purpose culture media

A

support the growth of large variety of organisms e.g. tryptic soy broth TSB

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2
Q

Enriched media

A

contains growth factors, vitamins and other essential nutrients to promote growth of fastidious organisms (organisms that cannot make nutrients needed so they have to be added to media).

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3
Q

Complex media

A

contains extracts and digests of yeasts, meats or plants, the precise composition of the media is unknown e.g. tryptic soy broth and brain heart infusion

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4
Q

Selective media

A

media that inhibits the growth of unwanted organisms and provides nutrients needed for organism and reduces competition.

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5
Q

Enrichment cultures

A

these foster the preferential growth of an microorganism that represents a fraction of the organisms present in an inoculum.

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6
Q

Differential media

A

these media change colour of the colonies of bacteria or the media depending on the organism present. this is as a result of interactions between bacterial enzymes with differential substrates in the media e.g. MacConkey Agar

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7
Q

LB agar

A

this is a general purpose medium that is suitable for the culture of many heterotrophic bacteria.

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8
Q

Heterotrophic bacteria

A

these require a supply of organic compounds in their environment.

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9
Q

Fresh blood agar

A

this provides additional nutrients that improve the growth of many bacteria, notably human commensals and pathogens. sometimes known as an indicator medium since it reveals the ability of some microbes to alter the appearance of blood.

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10
Q

MacConkey agar

A

this is a selective medium that will inhibit the growth of many organisms. it is also a differential medium and distinguishes between bacteria that can ferment lactose and those which cannot.

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11
Q

How to make culture media

A

Squirt ethanol over spatula to scoop out culture media
Weigh out 2 grams of dehydrated culture media on pan balance in a weight boat
Squirt ethanol over spatula for next user
Put dehydrated culture media into a conical flask
Add 200ml deionised water to conical flask with a bung on top and autoclave tape over bung and label bottle
• Put media in the autoclave to be sterilised

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12
Q

axenic culture

A

pure culture

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13
Q

problems with liquid based cultures

A

can only obtain pure cultures via successive dilutions which were tedious and prone to contamination.
only the commonest components of the culture would be recovered.

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14
Q

problems of solid gelatine media cultures

A

gelatine melts below 37C which is the optimum temp for isolation of human pathogens. also many bacteria digest gelatine causing medium to liquefy.

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15
Q

what is a Colony Forming Unit CFU

A

groups of cells giving ride to a single colony

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16
Q

Pour Plate Method

A

bacterial sample mixed with warm agar 45C - 50C
sample poured onto sterile plate
sample swirled to mix allowed to solidify
plate incubated until bacterial colonies grow

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17
Q

Spread plate method

A

sample (0.1 mL) poured onto solid medium
spread sample evenly over the surface
plate incubated until colonies grow on surface of medium

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18
Q

Streak plate method

A

Flame the loop and wire and streak a loopful of broth in a small section of agar.
Reflame the loop and cool it.
Streak next section along to spread the original inoculum over more of the agar.
Reflame the loop and cool it.
Streak next section.
Reflame the loop and cool it.
Streak at next section.
Label the plate and incubate it inverted.

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19
Q

Why is aseptic technique important?

A

to minimise the risk of contaminating your cultures with extraneous organisms, and prevent you from contaminating yourself with the cultures you are working on. this is particularly important when working with potential pathogens.

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20
Q

Chromophores

A

coloured biological molecules all have alternating single and double bonds causing them to be able to absorb light at different wavelengths

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21
Q

Beer Lambert Law

A

absorbance is directly proportional to concentration (c) and distance light travels through the solution (l). (E = molar absorbance coefficient)

A = Ecl

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22
Q

calculate percentage error

A

((observed - expected) / expected) X 100

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23
Q

calculate molarity (M)

A

Moles of solute (mol) / litres of solution (L)

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24
Q

Generation time

A

the time between the same points of the life cycle in two successive generations.

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25
Q

Doubling time

A

the time it takes for the population to double through one round of binary fission.

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26
Q

calculating number of cells

A

number of initial cells X 2(number of generations)

Nn = N0 2n
Nn = number of cells at any generation 
n = number of generations
N0 = number of initial cells
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27
Q

Culture density

A

the number of cells per unit volume

28
Q

intrinsic growth rate

A

the generation time under specific growth conditions (nutrients, temperature, pH) is genetically determined

29
Q

bacteria enumeration situations

A

In monitoring the microbial quality of drinking water
assessing the level of bacterial contamination in operating theatres
in quality control in the food and pharmaceutical industries
the numbers of microbes present may be of prime importance.

30
Q

how is Total cell count determined

A

is usually determined using a calibrated counting chamber, examined microscopically, or with the aid of a Coulter Counter,.

31
Q

issues with total cell count

A

The microscopic counting method is only suitable for counting relatively large numbers of bacteria, and it is not possible to differentiate viable and dead bacterial cells. Motile organisms require immobilisation.

32
Q

viable cell counts

A

measures the total number of living cells in a sample

33
Q

calculate total viable count

A

10 X (colonies per plate) X (dilution factor)

34
Q

spiral plater

A

is a semi-automated device. A stylus delivers a liquid inoculum onto the surface of a rotating plate in the shape of a spiral. As the inoculum is applied, the sample is being accurately diluted. The more bacteria there are in a sample; the further along the spiral growth will occur.

35
Q

Calculate the number of doublings

A

Number of doublings =log10(xt) - log10 (x0) / log10 (2)

xt= CFU/ml at the end of log phase
x0= CFU at the start of log phase
Log10(2) is 0.301

36
Q

calculate growth rate

A

number of doublings / time taken to grow that number of cells

37
Q

calculate doubling time

A

1 / growth rate

38
Q

Biuret assay

A

Low sensitivity 1-20 mg/mL
time taken = rapid 10 min
principle = peptide bonds + alkaline Cu2+ = purple complex
Interferences = Good’s buffers, Tris, some amino acids
comments = fast but not sensitive, similar colour with all proteins

39
Q

Lowry assay

A

sensitivity = high 5ug/mL
time taken = slow 40-60 min
principle = 1. biuret reaction 2. reduction of phosphomolybdate-phosphotungstate by Tyr and Trp
Interferences = ammonium sulphate, glycine, Good’s buffers, mercaptans
comments = time consuming, colour varies with proteins, critical timing of procedure

40
Q

Bradford assay

A

sensitivity = high 1 ug/mL
time taken = rapid 15 min
principle = (lamda)max of Coomassie dye shifts from 465 nm to 595 nm when protein-bound
Interferences = strongly basic, Triton X-100 and SDS detergents
comments = excellent method, only slight interferences, stable colour which varies with proteins, reagents commercially available

41
Q

BCA assay

A

sensitivity = high 1 ug/mL - 1 mg/mL
time taken = slow 30 min
principle = 1. conversion of Cu2+ to Cu1+ 2. copper complex with BCA (lamda)max 562nm
interferences = EDTA, dithiothreitolammonium sulphate
comments = compatible with detergents, reagents commercially available

42
Q

Spectrophotometric assay

A

sensitivity = moderate 50-100 ug/mL
time taken = rapid 5 min
principle = absorption of 280 nm light by Tyr and Try residues
Interferences = purines, pyrimidines, nucleic acids
comments = useful for monitoring column eluates. nucleic acid absorption can be corrected for

43
Q

How to change amount of light hitting specimen in microscope

A

adjusted by opening or closing a diaphragm between the condenser and the specimen. also using the rheostat, a dimmer switch that controls the intensity of the illuminator.

44
Q

why is a drop of oil used on a microscope sometimes?

A

air scatters the light before it enters the lens. a drop of oil can be placed to fill the gap of air using a oil immersion lens, as it has a similar refractive index to glass so it increases the maximum angle at which light leaving the specimen can strike the lens. this increases the light collected and therefore the resolution

45
Q

what is darkfield microscopy?

A

A small, opaque disk is placed between the illuminator and the condenser lens. The opaque light stop blocks most of the light from the illuminator as it passes through the condenser on its way to the objective lens, producing a hollow cone of light that is focused on the specimen. The only light that reaches the objective is light that has been refracted or reflected by structures in the specimen. The resulting image typically shows bright objects on a dark background.

46
Q

pros of darkfield microscopy

A

high contrast images, high resolution, does not need stains which could kill specimen so live specimens can be used.

47
Q

Phase contrast microscopy

A

uses refraction and inference caused by structures in the specimen to make high contrast, high resolution images.

48
Q

how does phase contrast microscopy work?

A

an annular stop produces a hollow cone of light that is focused on the specimen before reaching the objective lens.
The objective contains a phase plate containing a phase ring. so, light traveling directly from the illuminator passes through the phase ring while light refracted or reflected by the specimen passes through the plate.
This causes waves traveling through the ring to be about one-half of a wavelength out of phase with those passing through the plate.
Because waves have peaks and troughs, they can add together or cancel each other out. When the wavelengths are out of phase, wave troughs will cancel out wave peaks, which is called destructive interference.
Structures that refract light then appear dark against a bright background of only unrefracted light.

49
Q

light source and field iris

A

The light source provides the illumination for the microscope, which gets channeled through the condenser, the slide on the stage, the objective lens, and the eyepiece. This is referred to as the light path.

The field iris provides one way in which the intensity of light coming form the light source can be controlled. Closing down the field iris also helps us to set the focus and centering of the condenser when setting up the microscope.

50
Q

Condenser and aperture iris

A

The condenser collects light emitted from the light source and focus it onto the sample on the stage. The condenser ensures sufficient light is available to allow for the maximal resolution to be achieved at each magnification.

The aperture iris is another mechanism by which light intensity can be controlled.

51
Q

Stage and Objective lens

A

Slides for viewing under the microscope are placed on the microscope stage, within the slide holder.

The objective lenses (located above the stage) are on a carousel, and can be rotated into the light path of the microscope. This allows us to alter the magnification we use to view our slide specimens.

52
Q

Focus (stage height controller)

A

controls height of the stage and so the focus of the image

53
Q

X and Y axis controller

A

this moves the slide holder along the x and y axis

54
Q

Gram positive

A

thick peptidoglycan layer stained purple or blue

55
Q

Gram negative

A

thin peptidoglycan layer between the cytoplasmic membrane and the outer membrane. it does not retain the stain and appear pink or red

56
Q

steps of a gram stain

A

crystal violet added
iodine added
ethanol or acetone/ethanol solution added as a decolourising agent
counterstain usually safranin added

57
Q

autoclave temp and pressure

A

121C and 1 bar pressure

58
Q

Thermal death point

A

the temperature at which no organisms can survive in a broth culture usually after 10 - 20 minutes

59
Q

obligate aerobes

A

bacteria that depend on oxygen for survival

60
Q

obligate anaerobes

A

bacteria that are killed or inhibited by oxygen

61
Q

facultative anaerobes

A

do not require oxygen to grow but grow better in its presence

62
Q

microaerophiles

A

bacteria that are damaged by normal atmospheric levels of oxygen but do require a small amount (5%) in order to grow

63
Q

aerotolerant anaerobes

A

bacteria indifferent to the presence of oxygen

64
Q

psychrophiles

A

bacteria typically associated with polar regions, grow at 0C and have an optimum temp of below 20C

65
Q

Mesophiles

A

bacteria that grow between 15C - 45C. most are human pathogens and have an optimum of 37C

66
Q

Thermophiles

A

bacteria that can grow above 45C . often the optimum is between 55C - 65C

Biological Sciences (4 decks)

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