Workshop Lab Semester 1 Weeks 1-4

Question 1

All purpose culture media

Answer 1

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

Question 2

Enriched media

Answer 2

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).

Question 3

Complex media

Answer 3

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

Question 4

Selective media

Answer 4

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

Question 5

Enrichment cultures

Answer 5

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

Question 6

Differential media

Answer 6

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

Question 7

LB agar

Answer 7

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

Question 8

Heterotrophic bacteria

Answer 8

these require a supply of organic compounds in their environment.

Question 9

Fresh blood agar

Answer 9

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.

Question 10

MacConkey agar

Answer 10

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.

Question 11

How to make culture media

Answer 11

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

Question 12

axenic culture

Answer 12

pure culture

Question 13

problems with liquid based cultures

Answer 13

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.

Question 14

problems of solid gelatine media cultures

Answer 14

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

Question 15

what is a Colony Forming Unit CFU

Answer 15

groups of cells giving ride to a single colony

Question 16

Pour Plate Method

Answer 16

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

Question 17

Spread plate method

Answer 17

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

Question 18

Streak plate method

Answer 18

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.

Question 19

Why is aseptic technique important?

Answer 19

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.

Question 20

Chromophores

Answer 20

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

Question 21

Beer Lambert Law

Answer 21

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

A = Ecl

Question 22

calculate percentage error

Answer 22

((observed - expected) / expected) X 100

Question 23

calculate molarity (M)

Answer 23

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

Question 24

Generation time

Answer 24

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

Question 25

Doubling time

Answer 25

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

Question 26

calculating number of cells

Answer 26

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 ```

Question 27

Culture density

Answer 27

the number of cells per unit volume

Question 28

intrinsic growth rate

Answer 28

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

Question 29

bacteria enumeration situations

Answer 29

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.

Question 30

how is Total cell count determined

Answer 30

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

Question 31

issues with total cell count

Answer 31

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.

Question 32

viable cell counts

Answer 32

measures the total number of living cells in a sample

Question 33

calculate total viable count

Answer 33

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

Question 34

spiral plater

Answer 34

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.

Question 35

Calculate the number of doublings

Answer 35

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

Question 36

calculate growth rate

Answer 36

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

Question 37

calculate doubling time

Answer 37

1 / growth rate

Question 38

Biuret assay

Answer 38

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

Question 39

Lowry assay

Answer 39

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

Question 40

Bradford assay

Answer 40

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

Question 41

BCA assay

Answer 41

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

Question 42

Spectrophotometric assay

Answer 42

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

Question 43

How to change amount of light hitting specimen in microscope

Answer 43

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.

Question 44

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

Answer 44

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

Question 45

what is darkfield microscopy?

Answer 45

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.

Question 46

pros of darkfield microscopy

Answer 46

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

Question 47

Phase contrast microscopy

Answer 47

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

Question 48

how does phase contrast microscopy work?

Answer 48

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.

Question 49

light source and field iris

Answer 49

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.

Question 50

Condenser and aperture iris

Answer 50

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.

Question 51

Stage and Objective lens

Answer 51

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.

Question 52

Focus (stage height controller)

Answer 52

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

Question 53

X and Y axis controller

Answer 53

this moves the slide holder along the x and y axis

Question 54

Gram positive

Answer 54

thick peptidoglycan layer stained purple or blue

Question 55

Gram negative

Answer 55

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

Question 56

steps of a gram stain

Answer 56

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

Question 57

autoclave temp and pressure

Answer 57

121C and 1 bar pressure

Question 58

Thermal death point

Answer 58

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

Question 59

obligate aerobes

Answer 59

bacteria that depend on oxygen for survival

Question 60

obligate anaerobes

Answer 60

bacteria that are killed or inhibited by oxygen

Question 61

facultative anaerobes

Answer 61

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

Question 62

microaerophiles

Answer 62

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

Question 63

aerotolerant anaerobes

Answer 63

bacteria indifferent to the presence of oxygen

Question 64

psychrophiles

Answer 64

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

Question 65

Mesophiles

Answer 65

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

Question 66

Thermophiles

Answer 66

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