Lab Midterm Lecture Info Flashcards
first person to observe microbes, including bacteria, which he called “animalcules”
Antonie van Leeuwenhoek, 1600s
the use of any kind of microscope that uses visible light to observe specimens
light microscopy/compound microscopy
see living organisms, motility, bright objects on a dark background
dark-field microscopy
blocks most of the light from the illuminator in dark field microscopy
opaque disk
the only light that reaches the objective in dark field microscopy
refracted or reflected light by structures in the specimen
causes syphilis
treponema pallidum
two sets of light- one directly from the light source, one from light that is reflected or diffracted from a structure in the specimen
phase contrast microscopy
structures that differ in features such as ___ ___ will differ in levels of darkness; phase microscopy
refractive index
example- 40x objective lens
a. E- type apochromatic lens
b. 40x magnification
c. numerical aperture of 0.65
d. 160 mm mechanical tube length
e. 0.17 mm thickness cover slip
important markings on a light microscope objective lense
___ lenses are made in such a way that chromatic aberration is reduced to a minimum
apochromatic
describes the capacity of a microscope to enlarge an image
-objective and ocular
magnification
the ability to distinguish two adjacent objects as distinct and separate
resolution
light-gathering ability of the objective lense
numerical aperture
__ wavelength = better resolution
shorter wavelength
limit of resolution for a light microscope is about __ um
0.2
objects closer together than this value cannot be resolved as distinct and separate
limit of resolution
magnification of ocular lense
10x
D = wavelength / NAcondenser + NAobjective
formula for calculating the actual limit of resolution for a microscope
has the same refractive index as glass
immersion oil refractive index
increases the maximum angle at which light leaving the specimen can strike the lense
immersion oil
mostly UV or blue light
light source of fluorescent microscopy
uses an electron beam to create an image, with electromagnets acting as lenses
electron microscopy
image generated using flurescence
fluorescent microscopy
uses electron beams to observe small, thin specimens such as tissue sections and sub-cellular structures
transmission electron microscope (TEM)
uses electron beams to visualize surface 3D surface details of specimens
scanning electron microscope (SEM)
used to clean all lenses
dry, clean lens paper
used to remove oil from the stage
ethanol
-low-power objective in position and body tube lowered completely
-centered mechanical stage
proper set up of microscope
coil the electric cord around the body tube and the stage
proper cord position during microscope transfer
three types of bacterial morphology
cocci, bacilli, spiral
example of diplococci
streptococcus pneumonia, Enterococcus
example of cocci clusters
staphylococcus aureus
example of cocci chains
streptococcus pyogenes
example of flagellate rods
salmonella typhi
example of bacilli chains
bacillus anthracis
example of spore formers
clostridium botulinium
example of spirochetes
treponema pallidum morphology
example of spirilla
helicobacter pylori
example of vibrios- aka curved rods
vibrio cholera
will occur if a culture plate is left open on a lab bench
microorganisms will contaminate
practices and procedures to prevent contamination from pathogens and minimize the risk of infection
aseptic technique
6 inches
stay within ___ inches of bunsen burner to minimize contamination
a liquid medium
broth
usually made with 1.0 % - 2.0% agar in plates or tubes
solid medium
usually made with 0.3%-0.5% agar in plates or tubes
semi-solid medium
refers to cultivating and growing microorganisms in the lab on various types of media
culturing
-solidifying agent
-allow surface growth or restrict mobility
-grow bacteria over a range of temperatures
-liquifies at 100 C and solidifies at ~42C
agar
organism, name, section number, date
tube labels
how to sterilize an inoculating loop/needle
-hold in blue cone of flame at 45 degree angle
-let cool before transfer
-re-flame when finished and place upright back in block container
considered contaminated at all times
wire holder of inoculating loop/needle
color of inoculating needle/loop for sterilizing in flame
orange/red incandescence
-abundance of growth
-pigmentation
-optical characteristics
-form
microbe culture characteristics of agar slants
-growth type and distance from stab
-pigmentation or appearance
microbe culture characteristics seen in agar deep tubes
-abundance of growth
-type of growth
microbe culture characteristics seen in broth medium
transfer from agar slant to sterile broth tube
use loop
from broth stock to sterile agar deep tube
use needle and stab inoculation
from broth stick to sterile agar slant tube
use loop and inoculate surface of slant
incubation temperature of bacillus cereus and Serratia marcescens
25 degrees C for 24 to 48 hours, exercise 2
incubation temperature for staphylococcus epidermidis and Pseudomonas aeruginosa
37 degrees C for 24 to 48 hours, exercise 2
bacteria (pure or mixed culture) are diluted until single cells are separated from one another/isolation of distinct colonies
streak plate definition (4 quadrant streak)
-to grow into isolated pure colonies/determine purity of culture
-color, morphology and other physical characteristics
-quick first step in the identification of the bacteria being studied
purposes of streak plate
heavy confluent growth, light growth, discrete colonies
types of growth on streak plate
streak plate but with 3 sections instead of 4
t-streak
- sterilize loop
- touch loop to bacterial culture (broth or colony)
- streak heavy in one quadrant
- flame loop
- streak in and out of 1st quadrant a few times into 2nd quadrant then only streak in 2nd quadrant to fill
- repeat steps for the 3rd and 4th quadrants
- when done, sterilize loop
procedural steps for streak plate- isolating discrete bacterial colonies
-form
-elevation
-pigmentation/color
-size
-optical properties
microbe culture characteristics of agar plates
colonies are mucoid, raised, and shiny
Klebsiella pneumoniae agar plate properties
sample is pipetted onto surface of agar plate, sample is spread evenly over surface using sterile glass spreader, surface colonies seen
spread-plate method
sample is pipetted into sterile plate, sterile medium is added and mixed well, surface and sub-surface colonies seen
pour-plate method
24 to 48 hour nutrient broth cultures of a mixture of E. coli and B. subtilis
cultures for exercise 3
two trypticase soy (TS) plates per student
media used for exercise 3
incubation temperature for exercise
37 degrees celcius
practice aseptic technique to transfer cultures
-agar slant to terile broth tube (loop)
-broth to agar deep tube (needle and stab)
-broth to agar slant (loop surface)
exercise 2 culture transfer
perform two 3- or 4-quadrant streak plates and two spread plates using a mixed organism broth culture
exercise 3- isolating distinct colonies
picking up a single isolated colony
pure culture
to prepare a stock culture of an organism using isolates from the mixed cultures prepared on the agar streak plate and or the spread plate in exercise 3
purpose of exercise 4- preparation of pure cultures
solution consisting of a solvent (usually water or ethanol) and a colored molecule (often a benzene derivative)- the chromogen
stains
positive chromogen, stains cell
basic stain
negative chromogen, background is stained
acidic stain
auxochrome
provides covalent or ionic bonds in stain
colored compound, benzene (colorless) and chromophore (imparts color)
chromogen
developed the gram stain
Hans Christian Gram 1884
appear purple after staining
gram-positive bacteria
appear pink after staining
gram negative bacteria
differences in cell wall structure
reason for color difference in gram stain
has thick cell wall
gram-positive bacteria structure
has thin cell wall, with outer membrane
gram-negative bacteria structure
- primary stain- crystal violet
- mordant- iodine
- decolorizing agent- alcohol-acetone
- counterstain- safranin
order of application for gram stain
appears colorless after decolorizing agent
gram-negative cells
contains notable human pathogens such as M. tuberculosis, M. leprae
genus Mycobacterium
gram-positive bacteria that are acid-fast because of the waxy mycolic acid in their cell walls
mycobacteria
detects the presence of cell walls rich in mycolic acid
acid-fast staining protocol (Ziehl-Neelsen Method)
stains everything strongly in acid-fast staining
carbolfuchsin
decolorizing agent in acid-fast staining
acid alcohol
counterstain in acid-fast staining
-stains non acid-fast cells blue
methylene blue
structures that protect the bacterial genome in a dormant state when environmental conditions are unfavorable
endospore
bacillus and clostridium
endospore-forming, gram-positive bacteria genera
the process by which vegetative cells transform into endospores
sporulation
staining detects endospores
Schaeffer-Fulton method
malachite green and carbolfuchsin
need steam to enter cells for staining
- primary stain- malachite green
- spore retains malachite green while bacterias structures loose the stain- spores resist decolorization with water
- counterstain- safranin
process of spore staining- Schaeffer-Fulton Method
non-specific media configured to culture a wide range of microorganisms without many restrictions
general growth media
designed to suppress the growth of unwanted bacteria and encourage the growth of the desired microbes
selective media
make it easier to distinguish colonies of the desired organism from other colonies growing on the same plate
differential media
Nutrient agar and Luria-Bertani (LB) Broth
examples of general growth media
similar to selective media but designed to increase numbers of desired microbes to detectable levels
enrichment
both selective and differential for staphylococcus aureus
mannitol salt agar
high salt concentration (7.5% NaCl) inhibits growth of most bacteria except staphylococci
what makes MSA selective for staphylococci
carbohydrate mannitol is fermented by S. aureus, resulting in acidic end products- turns phenol red indicator yellow around growth
MSA differential reaction of staphylococcus aureus
microbiologists often use this to identify bacterial species that destroy red blood cells
blood agar
alpha (a) hemolysis
partial/incomplete lysis of RBC, zone of partial clearing = green halo around colonies
beta (B) hemolysis
complete lysis of RBC, complete zone of clearing around colonies
gamma (Y) hemolysis
no lysis of RBCs, no clearing of medium surrounding colonies, no color change
tends to be used to recover fastidious bacteria, often Streptococcus species (pathogens)
blood agar bacterial species
blood agar is a ___ media, bacteria distinguished by ability to cause hemolysis
differential media (hemolysis)
selective and differential medium containing lactose, bile salts, neutral red, and crystal violet
MacConkey Agar
interferes with the growth of many gram-positive bacteria and FAVORS gram-negative bacterial growth, especially Enterobacteriaceae
MacConkey agar favorable growth
named enterics, reside in the intestine, are adapted to the presence of bile salts
Enterobacteriaceae
ph indicator in MacConkey agar, colorless above a pH of 6.8 and red at a pH below 6.8
- acid accumulating from lactose fermentation turns dye red
neutral red dye
-used for the isolation and identification fo fecal coliform bacteria
-sugars act as fermentable substrates, which yield acid by-products
-dyes inhibit growth of gram-positive bacteria and act as pH indicators (only used to see gram-negative)
Eosin Methylene Blue Agar
eosin and methylene blue inhibit growth of gram-positive bacteria
why eosin methylene blue agar is selective
distinguishes gram-negative bacteria that can ferment lactose from those that cannot
why eosin methylene blue agar is differential
lactose fermenters in eosin methylene blue agar
produce dark colonies- metallic green shade
non lactose fermenters in eosin methylene blue agar
produce opaque or translucent colonies
example of lactose fermenter
Escherichia. coli
small amounts of acid production in eosin methylene blue agar
results in pink coloration of the growth
an undefined, selective medium that allows growth of gram-positive organisms and stops or inhibits growth of most gram-negative orgamisms
phenylethyl alcohol agar
false negative results and low colony counts
viable but non-culturable (VBMN)
bacterial species that will grow within a temperature range of -5°C to 20°C
- all will grow between 0° and 5°
psychrophiles
bacteria species that will grow within a temperature range of 20°C to 45°C
-all can grow at human body temperatures (37°C) and are unable to grow above 45°C
-three distinct groups
mesophiles
optimal growth at 20-30°C
-mesophile
plant sacrophytes
optimum growth temperature at 35-40°C
-mesophile
organisms that grow in warm blooded hosts
optimum growth at 20-40°C but are capable of growing at 0°, typically found in soil and water habitats in temperate regions
-mesophile
psychrotolerant
bacterial species that will grow at 35°C and above
-two groups exist
thermophiles
thermophiles that will grow at 37°C but grow optimally at 45-60°C
facultative thermophiles
thermophiles that will grow only at temperatures above 50°C, optimum growth above 60°C
obligate thermophiles
detection of gas accumulation
air bubble in durham tube (within a culture tube)
enzyme that protects cell from toxic H2O2- hydrogen peroxide
catalase and peroxidase
enzyme that protects cell from toxic O2^- superoxide
superoxidedismutase
has no enzymes to protect against toxic oxygen- cannot grow in its presence
strict anaerobes
-requires the presence of atmospheric oxygen for growth
-their enzymatic needs to use oxygen as the final electron acceptor
aerobes
microaerophiles
-require limited amounts of atmospheric oxygen for growth
-excess oxygen blocks the activities of their oxidative enzymes and results in death
require the absence of free oxygen for growth because the require the presence of molecules other than oxygen to act as the final electron acceptor
-presence of oxygen is lethal
obligate anaerobes
fermentative organisms that do not use oxygen as a final electron acceptor
-produce enzyme so they can survive in the presence of toxic oxygen
aerotolerant anaerobes
can grow in the presence or absence of free oxygen
-preferentially use oxygen for aerobic respiration but can perform cellular respiration anaerobically if necessary
-can use nitrates or sulfates as final hydrogen acceptors or use a fermentative pathway
facultative anaerobes
proof of life/viable counts
show that an organism can replicate and form colonies on an agar plate
serial 10 fold dilutions
allow estimation of the number of live organisms in the initial sample
pros of serial dilution
allow for colony isolation, quantifies only live cells, controls for mixed cultures
cons of serial dilution
takes longer (incubation time), user error, material use
less than 30 colonies, TFTC
too few to count
more than 300 colonies, TNTC
too numerous to count
colony forming units / milliliter of initial culture
CFU/mL
how to present results of serial dilution viable counts
CFU calculation formula
(number of colonies on plate) x (reciprocal of dilution of sample)
standard CFU formula
(colony count on agar plate) / (total dilution of tube) x (amount plated)
bacteria normally reproduce by __ __
binary fission- how bacteria reproduce
intense activity preparing for population growth, but no increase in population
lag phase of the growth curve
logarithmic, or exponential, increase in population
log phase of the growth curve
period of equilibrium; microbial deaths balance production of new cells
stationary phase of the growth curve
population is decreasing at a logarithmic rate
death phase of the growth curve
the logarithmic growth in the log phase is due to reproduction by ___ ___ (bacteria) or __ (yeast)
binary fission, mitosis
the generation time of a culture
the growth rate of a culture, determined by the number of cells at several time points, only calculated during the log phase
equation of generation time
g = t/n
g - generation time (minutes)
t - time of exponential growth
n - the number of generations
turbidity- optical density
- 600 nm light source
detects “cloudiness” or transmittance through a medium
pros of optical density
quick and easy
cons of optical density
do not distinguish between live and dead cells, contamination not taken into account, does not allow for isolation of colonies
formula for n
(log N - log N0) / log 2
axes of graph converting optical density to cell number (original graph)
x- optical density
y- cell number
axes of graph used to determine generation time (new graph)
x- time
y- cell number
polymer of collagen that makes up connective tissues, liquid above 25°C
gelatin
break down collagen
-nutrient acquisition, virulence
gelatinases (collagenases)
liquefaction after growth followed by refrigeration indicates hydrolysis
gelatin hydrolysis test
iodine is used to detect the presence of starch, hydrolysis revealed as a clear zone around bacterial growth
amylase/starch hydrolysis test
tributyrin agar, lipase-positive organisms produce clear zone around growth
lipid hydrolysis
converts 1 molecule of glucose to 2 molecules of pyruvate
glycolysis
Embden-Meyerhof pathway
molecular oxygen as the final electron acceptor (more ATP generated)
aerobic cellular respiration
inorganic ions rather than oxygen are final electron acceptor
anaerobic cellular respiration
doesn’t require oxygen and organic substrate is the final electron acceptor
fermentation
may cause acid and gas
carbohydrate fermentation
indicator of acid production (yellow = acid)
phenol red indicator
indicator of presence of air bubble
durham tube
indicates that an organism can metabolize sugar in the tube
color change red to yellow, acid production
fermentation and gas production
color change and bubble in the durham tube
color change to a dark pinkish-red for carbohydrate fermentation
indicates a basic or alkaline metabolic product due to utilization of the peptone rather than the sugar
reacts with indole to produce red/pink color
Kovac’s reagent
tryptophanase
enzyme used to identify bacteria that produce indole
produced by certain enterobacteriaceae by two pathways
H2S (Hydrogen sulfide gas)
combines with hydrogen sulfide gas to form black sulfide precipitate
FeSO4 (ferrous ammonium sulfate)
a positive result for __ is indicated when radiating outward from the central stab; a negative result shows only along the stab line
motility
all ferment glucose to organic acids (the acid varies)
enteric microorganisms
differentiates between E. coli and K. aerogenes final end products
MR-VP tests
determine ability to oxidize glucose to make acid end products (E. coli)- makes red color
methyl red test
K. aerogenes converts acids to acetylmethylcarbinol raising the pH brining color back to yellow
-barritt’s reagent turns pink with acetylmethylcarbinol
voges proskauer test
determine the ability of an organism to use the enzyme citrase to use citrate as a sole carbon source
citrate IMViC test
growth on the slant and blue color
means citrate was utilized as a carbon source
differentiate organisms based on their ability to hydrolyze urea with the enzyme urease
urease test
urinary tract pathogens from the genus __ may be distinguished from other enteric bacteria by their rapid urease activity
Proteus
broth tube turns bright pink
positive urease test
fissures or cracks in the clot of litmus tube
gas production in litmus tube
acid clots solidify the medium and can appear __ or __ with a pink band at the top depending on the oxidation-reduction status of litmus
pink, white
lactose fermentation acidifies the medium and turns the litmus pink
pink in litmus milk test
reduced litmus is white
white color in litmus test
oxidized litmus is purple
purple in litmus test
alkaline reaction for litmus test
blue/purple medium or blue band at the top
stormy fermentation
heavy gas production that breaks up the clot
digestion of peptone
milk protein completely digested, clear to brown fluid
acid clot formation
proteolysis
enzyme catalase
degrades hydrogen peroxide
performed on a glass slide, colony + hydrogen peroxide - bubbles form
positive catalase test
uses a chromogenic reducing agent as an indicator to detect bacteria that produce cytochrome oxidase
oxidase test
indicator in oxidase test
- donates electrons to cytochrome oxidase, becomes self oxidized changing from light pink reduced to a dark maroon almost black oxidized compound
p-aminodimethylalanine oxalate
