Microbio Lab Test 2

Helpfulness: 0
Set Details Share
created 4 years ago by ahudson4
36 views
updated 4 years ago by ahudson4
Subjects:
microbio lab
show moreless
Page to share:
Embed this setcancel
COPY
code changes based on your size selection
Size:
X
Show:
1

Complex medium

contains amino acids, nucleotide bases, vitamins, etc. required for growth--exact composition of growth factors unknown

2

Defined medium

Composed of specific components at specific amounts to support growth of known bacteria

3

Heterotrophs

Obtain carbon from organic compounds, like polysaccharides, carbs, amino acids, proteins, etc.

4

Autotrophs

Derive carbon from CO2 fixation

5

Chemoorganotroph

respiration or fermentation used to create energy, obtain carbon from organic compounts

6

Chemolithotroph

oxidize inorganic ions (nitrate, iron, etc.) to obtain energy to fix CO2

7

Photoautotroph

convert solar energy to chemical energy, derive carbon from fixing CO2

Contain photosynthetic pigments like chlorophyll, bacteriophyll

cyanobacteria, green and purple sulfur bacteria

8

Additional requirements for microbial growth (besides carbon and energy source)

Nitrogen, minerals, vitamins, growth factors, water

9

Endospore

resting stage when nutrients are exhausted; can germinate into vegetative cells when nutrients reintroduced

Resistant to heat, radiation, acid, chemicals, because of exosporium (protein coat)

Stained with malachite green, vegetative cells with safranin

Have v little H2O (10-30%), so more heat resistant

10

Microscopic counts

Diluted sample counted using a microscope and special slide with grids called Petroff-Hauser chamber

Amount of sample added is known

used to count bacteria in milk!

11

Petroff-Hauser chamber

gridded slide used in microscopic counts

12

Most Probable Number

Relies on relationship of some growth parameter to statistical probability

often used to test water quality

13

Standard Plate Count/ Viable Count

First, serially dilute bacteria

number of bacteria in original sample is determined by multiplying number of colonies by dilution factor

Not all colonies are single cell, so called colony forming unit (CFU) and counted between 30 and 300 for statistical validity

Biased, because only allows certain conditions and media (i.e. soil bacteria underrepresented in this method)

Counts only living cells!

14

Colony forming unit (CFU)

= (# colonies counted * dilution factor)/ volume plated

*dilution factor= reciprocal of dilution ratio

resulting unit=CFUs/ ml

15

Optical density/ absorbance

Indirect method of counting bacteria--measure turbidity in spectrophotometer

Measures living and dead cells

16

Using spectrophotometer

Spec measures % transmittance, A=log (1/T)

Set wavelength to 550 nm, zero machine, set %T to 0%

Calibrate! Use broth as blank, set %T to 100%, then read %T for each dilution

Standard curve for OD vs. CFU/ ml generated!

17

Reasons why E. Coli is good indicator bacteria for water quality

1) found in intestine, but not soil or water

2) easily identified through tests

2) survives for long time in water

18

Coliforms

Gram-negative, lactose-fermenting (produce acid and gas), non-endospore forming rods that are also facultative anaerobes

Intestinal bacteria

19

Presumptive test

Tests for lactose fermenting in coliforms

Calculate MPN in double strength and single strength lactose broths with Durham tubes, use chart

20

Confirmed test

Tests for lactose fermenting and gram-negative

Use after presumptive test to confirm water with gas pockets is unsafe to drink

Use Endo or Emb media

21

Emb (Eosine methylene blue) media

selective media that slightly inhibits the growth of gram positive bacteria (both dyes eosin and mb inhibit growth)

Differential media on which lactose fermenters turn purple-black, E. Coli (and E. aerogenes) turn metallic green

22

Endo agar

Lactose fermentsrs turn red; non-fermenters are clear

Contains fuchsin sulfur as indicator

23

Completed test

Performed after presumptive test and confirmed test

Tests for lactose-fermenting, gram-negative, non-endospore-forming rods

Use NA slants to grow bacteria, look at them on slides

24

Why do we care about microbial growth?

Microbiologists--want to maximize conditions for growth

Medical practitioners--want to limit growth to prevent disease

25

Obligate anaerobes

Must grow in O2!

Use O2 as final electron acceptor in ETC

26

Microaerophiles

Prefer O2 concentration between 2 and 10% (lower than atmospheric 20%)

perform respiration

27

Facultative anaerobes

Flexible metabolism

Respire when O2 present, ferment when O2 absent

Grow better when O2 present

28

Aerotolerant anaerobes/ obligate fermenters

tolerate O2, but don't require it

29

Obligate anaerobes

Can't grow in presence of O2

Use inorganic ions as terminal electron acceptors (like nitrate and sulfate)

Found in soil, cattle rumen, intestines

30

Reactive oxygen species (ROS)

By-product of respiration that damage DNA, proteins, coenzymes

Most microbes have catalase, superoxide dismutase to deal w/

Most strict anaerobes don't have catalase, superoxide dismutase; some have other methods to deal w/ ROS

31

Catalase

Degrades H2O2 into H2O and O2

Missing in aerotolerant anaerobes

32

Superoxide dismutase

converts superoxide anion to H2O2 and H2O

33

FTM (fluid thioglycollate)

Supports growth of aerobic and anaerobic bacteria

contains resazurin dye--turns pink in O2 presence

Thioglycollate (and cystine) react with O2 to form H2O, anaerobic conditions

34

Psychrophile

-5-20 degrees C

35

Pychrotroph

4 degrees C

36

Mesophile

20-40 degrees C

37

Thermophile

50-80 degrees C

38

Hyperthermophile

>80 degrees C

39

UV light

Causes pyrimidine dimers in DNA--covalent bonds between 2 adjacent thymine or cytosine molecules that prevent replication and transcription

40

SOS system

removes dimers and replaces pyrimidine molecules

Doesn't function properly if too many dimers present--> cell death

41

UV effectiveness

Depends on:

1) length of exposure time

2) Blocking (glass, plastic)

3) presence of endospores

42

Antiseptics

Kill/ inhibit microbes, not endospores--gentle enough for human skin

Alcohol kills bacteria by denaturing proteins and membranes

Also includes iodine, hydrogen peroxide

43

Disinfectancts

kill/ inhibit microbes on surfaces and objects

bleach, formaldehyde, phenol, oxidizing agents

Sterilants & sporocides kill spores, i.e. ethylene oxide gas

44

Photoheterotrophs

derive energy from photosynthesis

derive C from growth in organic molecules like succinate and glutamate

purple nonsulfur bacteria

45

Sources of nitrogen in media

Beef extract

Peptone

46

Minerals

cofactors in enzymatic reactions

integral parts if cytochromes, bacteriochlorophyll, vitamins

K, Na, Fe, Cu, Zn, etc.

47

Vitamins

Coenzymes in metabolism

niacin in NAD, flavin in FAD

48

Rules to follow in gram staining

1) cultures 16-18 hrs old-- if too old, gram-pos bacteria may turn gram-variable or gram-neg, giving erroneous results

2) thin smears! to allow decolorization to function properly; if too thick, smear can entrap stain, making gram-neg cells appear gram-pos

3) Decolorization=most important step! if leave alc on too long, remove dye-mordant complex from gram-pos cells, removing color

49

Schaefer-Fulton method

endospore staining technique

50

Peroxidase

degrades H2O2

51

Temperature

Enzymes have optimal temps; otherwise, denature

Affects cell membranes and transport; as temp decreases, transport decreases due to change in membrane fluidity

if temp too high, membrane lipids can be destroyed

52

Prodigiosin

red pigment produced by s. marcescens

antibiotic and cancer drug

produced at specific temperatures only

53

Spore resistance to UV

1) DNA protected by small acid-fast proteins that bind to DNA and alter conformation

2) spore photo-product generated by UV that functions as DNA repair mechanism during endospore germination

54

Teichoic acids

Found in gram-positive cell walls

Limit degradation of peptidoglycan by autolysins and lysozymes