Final exam review

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Final exam review
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Microscope anatomy
http//shs.westport.k12.ct.us/mjvl/biology/microsc
ope/microscope.htm
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Gram - bacteria

• Small peptidoglycan
• Are stained with crystal violet but decolorized
  with alcohol after which they pick up the red
  stain
• LPS on outer membrane ? toxic for host

http//www.cat.cc.md.us/courses/bio141/lecguide/un
it1/prostruct/toll/u1fig10b.html
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Gram bacteria

• Large, highly cross-linked petidoglycan
• No outer membrane

http//www.cat.cc.md.us/courses/bio141/lecguide/un
it1/prostruct/u1fig9b.html
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Bacteria shapes

http//ibri.org/RRs/RR051/51bacterialshapes-Merc.g
if
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Interpretation of results
Bacteria differ in their rate of decolorization
MIC 205L - LEC Gram Staining
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Mycobacteria structure

• Contain large amount of fatty waxes (mycolic
  acid) within their cell wall ? resist staining by
  ordinary methods
• Require a special stain for diagnostic ? Acid
  Fast stain.

http//www.med.yale.edu/labmed/casestudies/images/
cs4_mycolic_acid.jpg
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Acid Fast Stain

• Mycobacterium smegmatis (pink) ? acid fast retain
  the carbolfuchsin dye
• Micrococcus luteus (blue) ? not acid fast ?
  decolorized with acid-alcohol ? conterstained
  with methylen blue

http//www.bact.wisc.edu/Microtextbook/images/book
_3/chapter_3/3-17.jpg
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Endospore staining

• Spore green
• Vegetative cell red

http//www.arches.uga.edu/howie/MVC-052endoS.JPG
http//homepages.wmich.edu/rossbach/bios312/LabPr
ocedures/endospore.jpg
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Sterile techniques what can you do in the lab?

• Wash your hands
• Keep your bench clean
• Wear gloves
• Flame loop, neck of tube
• Keep cap facing down
• Work quickly albeit efficiently
• Limit talking when opening cultures

http//www.parentsguidecordblood.com/vita34cleanro
om50.jpg
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Culture media
Plate
Broth
Slant
Deep
http//student.ccbcmd.edu/courses/bio141/lecguide/
unit2/control/images/broth.jpg
http//www.mushmush.nl/images/methods/working_with
_agar/slant.jpg
http//82.43.123.182/globalplantclinic/images/Bact
eria_plate.jpg
http//student.ccbcmd.edu/courses/bio141/labmanua/
lab7/images/negmotility.JPG
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Bacteria motility

• Non motile bacteria will only be found at the
  site of inoculation
• Motile bacteria ? swim around go everywhere

http//www.bact.wisc.edu/bact100/Motility.jpg
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Uses

• Broth
• High concentration of bacteria
• Slant
• Space saving solid culture

• Plate
• Individual colonies
• Can be used to count bacteria
• Deep
• Look at motility oxygen requirement

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Pure vs mixed culture

• Pure originate from 1 bacteria strain
• All colonies look the same
• Mixed originate from many bacteria strains
• Colonies have different size/shape

http//smccd.net/accounts/case/biol240/streakplate
.html
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Streak plate technique

• Spread millions of cells over the surface
• Individual cells deposited at a distance from all
  others
• Divide forming distinct colonies
• Distinct colonies do not touch any other colonies
• Clone of a single bacteria ? pure culture

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Three streak plate technique

• Using a sterile loop take a loopful of your
  bacteria from the broth
• Streak a vertical line
• Then streak gently across section 1
• Zig-zag pattern until a 1/3 of the plate is
  covered
• Do not dig into the agar

http//www.rlc.dcccd.edu/MATHSCI/reynolds/MICRO/la
b_manual/streakplate2.jpg
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Isolated coloniesColony Forming Units CFU
http//faculty.mc3.edu/jearl/ML/ml-9.htm
http//www.bact.wisc.edu/themicrobialworld/Prop.ac
nes_colonies.jpg
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Counting bacteria

• We must have between 30 and 300 colonies on the
  plate
• Less than 30 might not be representative
• More than 300 very difficult to count
• Also we might not have isolated colonies

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CFU calculation example

• You count 46 colonies on your plate
• You put 1 ml of bacterial culture into 99 ml of
  saline and plated 0.1 ml
• Dilution 1/100
• CFU 46
• 1/100 0.1
• 46 100 10 46 000
• Dividing by 1/100 is the same as multiplying by
  100 0.1 1/10 Do not take amount plated in
  consideration twice

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Lab 7. Microbial growth

• During this lab you will learn how some physical
  factors can influence the growth of bacteria.
• Bacteria have preferences with regard to some
  physical factors
• Oxygen requirement
• Temperature
• Osmotic pressure

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Oxygen requirement
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Thyoglycollate broth

• 1. Strict aerobes grow only at the surface
• 2. Facultative anaerobes grow throughout the
  tube, but some do better at the surface
• 3. Aerotolerant grow throughout the tube
• 4. anaerobes grow in lower portion of the tube

http//www.bact.wisc.edu/Microtextbook/images/text
book/growth/thioglycollate.jpg
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Temperature requirement

• Bacteria can usually grow over a wide range of
  temperature
• But often have a temperature at which they grow
  best (divide more rapidly)
• Psychrophilic cold loving (
• Mesophilic middle loving (30-37oC)
• Thermophilic heat loving ( 50oC)

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Disinfectant

• Chemical used on surfaces of inanimate object to
  kill microbes
• Chlorine swimming pool
• Chloramine drinking water
• Alcohol lab bench, hospital. This is more
  effective if mixed with water (70 more effective
  than 95)

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Antiseptics

• Chemical used on living tissues to kill microbes
• Alcohol
• Iodine
• Hydrogen peroxide
• Some disinfectants are also antiseptics
• In surgery prefer to use aseptic (sterile)
  techniques instead of antiseptics

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Antibiotics

• Drugs that kill or inhibit the growth of some
  bacteria
• Bactericide (kill)
• Bacteriostatic (Inhibit growth)
• Target microbes with minimal side effects to the
  host
• Will also kill friendly host bacteria (such as
  the ones in digestive tract)

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Objective 1 Effectiveness of Antibiotics
Disk-diffusion technique

• Swab the entire surface of 1 MH (Mueller-Hinton)
  plate with 1 bacterial culture

• Staphylococcus aureus, Pseudomonas aeruginosa,
  Bacillus subtilis, or Escherichia coli Place 3
  paper disks containing antibiotics onto the MH
  plate. you use 3 different antibiotic per plate,
  3 disk total per plate
• Let the plate dry for a 5 minutes. So the disks
  adsorb to the agar. ? invert
• Incubate at 37C.

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Zone of inhibition

• resistant

intermediate
susceptible
http//dept.kent.edu/microbiology/htm/resist.jpg
http//www.bspp.org.uk/bsppnews/bsppnews38/byron.j
pg
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Objective 1 Carbohydrate fermentationA. Lactose
broth

• Escherichia coli
• Proteus vulgaris
• Positive result yellow color change with or
  without gas production
• Negative result no color change
• Gas production ? bubble in the durham tube

http//www2.austin.cc.tx.us/microbugz/images/26suc
.jpg
http//biosci.usc.edu/courses/2002-fall/documents/
bisc300-lab_Carbohydrate_Fermentation_Sucrose.jpg
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Objective 1 Carbohydrate fermentationB. OF test
(oxidation-fermentation)

• Contains a Glucose
• pH indicator bromothymol blue ? turn yellow
  under acidic condition
• Determine whether a bacterium has the enzymes
  necessary for the aerobic breakdown of glucose
  (oxidation) and/or for the fermentation of
  glucose (anaerobic).

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OF glucose test Use the needle
oil
1 no oxidation, no fermentation 3
Fermentation 2 oxidation but no fermentation
http//www.jlindquist.net/generalmicro/DMimages/ne
wglucof2.jpg
Escherichia coli Pseudomonas aeruginosa
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Objective 3 MR-VP

• Methyl red
• Identify bacteria that produce stable acids as
  end-products when fermenting glucose
• pH indicator added after incubation
• After 24 hours,Add Methyl Red 5 6 drops
• Positive Red
• Negative yellow

E. Coli E. Aerogenes
http//www.marietta.edu/spilatrs/biol202/labresul
ts/methyl20red.jpg
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Objective 3 MR-VP

• Voges-Proskauer
• Identify bacteria that produce butanediol as
  end-products of fermentation
• Add 15 drops of reagent 1
• Add 5 drops of reagent 2
• Color change can take up to 30 60 minutes
• Positive red color
• Negative yellow/translucent color

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Catalase test
24 hours later add hydrogen peroxide on the
colony
Bubble positive result
http//biology.ucok.edu/Microbiology/images/catala
se.jpg
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Objective 2 Indole Production

• Positive red ring at the top of the media
  (Indole present)
• Negative brown or yellow ring at the top of the
  media

http//medic.med.uth.tmc.edu/path/indole.jpg
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Objective 4 Lysine decarboxylation

• 1 Negative reaction No fermentation
• LDBPurple
• Controlpurple
• 2 positive reaction.
• LDB purple descarboxylation of lysine Control
  yellow fermentation of glucose.
• 3 Negative reaction.
• LDByellow fermentation of glucose, but
  lysine is not decarboxylated,
• Control yellow fermentation of glucose.

Lysine Decarboxylase Broth LDB
http//www.bact.wisc.edu/Microtextbook/index.php?m
oduleBookfuncdisplayarticleart_id269
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Objective 6 Differential vs Selective Media

• Selective media some bacteria grow some other
  dont
• Differential all bacteria grow but will have a
  different appearance depending of their
  biochemical characteristics
• TSA
• MSA
• Mac Conkey
• EMB

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Objective 6 TSA

• Trypticase Soya agar Nutritious medium for a
  growth of a huge variety of bacteria.
• No differential
• No selective

http//www.austincc.edu/microbugz/assets/images/MS
A.jpg
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Objective 6 MSA

• MSA Mannitol Salt Agar
• Selective medium for Gram positives. Inhibit Gram
  negatives.
• Differential mannitol fermenters
• Medium specific for staphylococcus ? contains
  7.5 salt which is inhibitory for most other
  bacteria.
• Contains the sugar mannitol and the pH indicator
  phenol red ? fermentation of mannitol ? acid
  produced ? media turn yellow

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Objective 6 MSA

• Positive Growth, yellow
• Negative No growth, no change of color

http//www.austincc.edu/microbugz/assets/images/MS
A.jpg
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Objective 6 EMB

• EMB Eosin Methylene Blue
• Selective medium for Gram negatives. Inhibit Gram
  positives.
• Differential medium for lactose fermenters
• Lactose fermenter will produce dark red colonies
• E. coli produce a characteristic green sheen

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Objective 6 EMB

• 1 E. coli ? green
• 2 no lactose fermentation
• 3 lactose fermentation
• 4 no growth

http//www.microchemlab.net/bact5.jpg
http//iws2.ccccd.edu/dcain/CCCCD20Micro/EMBplate
.jpg
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Hemolysis

• Some bacteria have the ability to break down red
  blood cells
• This characteristic can be used for bacteria
  identification
• Stretococcus spp.

http//gold.aecom.yu.edu/id/micro/hemolysisabg-72.
jpg
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Alpha hemolysis

• Incomplete hemolysis
• Greenish darkening of the agar under the colonies
• Displayed by Streptococcus pneumoniae
• Caused by peroxide produced by the bacteria

http//gold.aecom.yu.edu/id/micro/hemolysis.htm
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Beta hemolysis

• Complete hemolysis, giving a clear zone with a
  clean edge around the colony.
• Streptococcus pyogenes
• Caused by the presence of hemolysine

http//gold.aecom.yu.edu/id/micro/hemolysis.htm
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Gamma hemolysis

• No hemolysis
• No change in the blood agar around the colony
• Ex enteroccocus faecalis

http//gold.aecom.yu.edu/id/micro/hemolysis.htm
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Lab 14. Simulated Epidemiology
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Bacteria used for experiment
serratia
http//www.arches.uga.edu/dlg/pigmentml.jpg
http//www.health.qld.gov.au/EndoscopeReprocessing
/images/page_images/1317_serratia.jpg
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Eukaryote organisms

• Nuclei
• Membrane bound organelle
• Also differences in genetic material, replication
  etc
• No peptidoglycan

• Eukaryotehttp//www.windows.ucar.edu/earth/Life/im
  ages/celltypes.gif

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Filamentous fungi

• Multicellular organism
• Long branched filament called hyphae
• Hyphae aggregate to form a mass ? mycelium
• Aerobes

http//www.anselm.edu/homepage/jpitocch/genbios/31
-01-FungalMycelia-L.jpg
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Sabouraud agar

• Selective media
• Contain sugars and peptone
• Low pH (5) which is inhibitory for most other
  microorganisms
• Invented by a French Doctor (Dr. Sabouraud)
  specialist in scalp disease

http//www.bium.univ-paris5.fr/sfhd/img/gd/sabou.j
pg
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Reproduction
Conidiospores are not enclosed within a sac
Sporangiospores enclosed in a sac like head
called a sporangium
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Fermentation

• If acid produced? media turns yellow (pH
  indicator)
• If gas production, bubble get trapped in the
  small tube (Durham tube)

http//faculty.mc3.edu/jearl/ML/glucose.jpg
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MPN statistical table

• Tube that turn yellow and gas production? ,
  presumptive evidence for coliformes.
• Tube not yellow ? -
• (Tube without gas ?-

3 1 1
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MPN statistical table
75 is the MPN of coliforms per 100 mL of water