lab 2, 3, 4 Flashcards
agar
solidifies medium
relatively stable polysaccharide
wide variety of heterotrophic microbes will grow on the surface of a t-soy plate by absorbing the nutrients out of the solid plate beneath them
agar cant be degraded by most terrestrial microbes, so the plate remains solid even when colonies have consumed most of the other nutrients in the plate
to count bacteria perform a standard plate count
a bacterial culture or a sample of water or food is spread on an agar plate and the number of colonies is counted through a simple calculation, you can determine the titre of bacteria in the original sample
the titre or concentration of bacteria determined by a standard plate count is expressed as the number of colony forming units per ml or (cfu/ml)
to carry out standard plate count there are two techniques
pour plate
spread plate
serial dilutions
a bacterial culture may have billions of bacterial cells in it. to makes this number more manageable, we must dilute the original sample so that we can obtain a plate with a countable number of colonies
ten fold serial dilution
0.1
only use plate counts from…
30-300
TNTC
too numerous to count
streak plate technique
dilute the culture as you spread it around so that in the end, you have nicely isolated colonies
an isolated colongy is a
colony that is not touching any other colonies on the plate
pure culture
if all of your colonies appear exactly the same
inoculating loop
used to pic colonies off a plate or to obtain a drop of liquid culture
the wire heats up and cools down very quickly
inoculating wire
pick up very small amounts of bacteria
pick a colony from a plate that is located very close to another colony
and to inoculate stab tubes
media (lab manual page 36)
singular medium
nutrient source for growing bacteria
can be solid liquid and can be prepared in a variety of containers such as tubes , flasks or plates
pour plate technique is commonly used to enumerate bacteria in food samples
because it does an excellent job of isolating colonies
it is difficult to observe bacteria using a brightfield microscope without
staining first as most bacteria are colourless
stain is composed of salts of coloured compounds suspended in a liquid usually water
charged portion of the salt molecule, called the chromophore, can have either gram positive or gram negative charge
bacteria have a net negative charge on their outer surface
basic stain AKA cationic stain
positively charged chromophore these stains are attracted to the negatively charged surface of the cell and can be used for staining cell surfaces and structures -crystal violet -safranin -methylene blue
acidic stain or anionic
negatively charged chromophore
these stains are repelled by the negatively charged cell surface and can be used to stain the background of the slide in a process called negative staining
-nigrosin
-india ink
bacteria most common shapes are
cocci, bacilli
spirillia
not all bacteria have a characteristic arrangement
singular
or random
most rod shaped bacteria occur as singles but can look like pairs when they divide
Micrococcus luetus simple stain
very small cocci arranged in tetrads
gram positive
gram positives
have a negative charge on their surface because of teichoic acids embedded in the thick peptidoglycan layer
gram negatives
have a negative charge on their surface bc of the charged sugars and phosphates in the lipopolysaccharide component of the outer membrane
both gram negative and gram positives have
negatively charged surfaces
E. coli
gram negative
bacilli
singular
B. su
gram positive
bacilli
either singular or streptobacilli
s. ep
gram positive
coccus
staphylococci
why is alcohol destaining the differential step
bc gram positives and gram negatives will react differently to this treatment
this is the step that lets you see a difference between the two cell types
-ethanol shrinks the pores of the peptidoglycan layer in the gram positive bacteria, trapping the crystal violet iodine complex
-in gram negative the ethanol disrupts the outer membrane which allows the loosely trapped crystal violet molecules to be washed out of the thing peptidoglycan layer
safranin is a basic counter stain
a counterstain is a stain that sticks to structures not bound by a primary stain
- when counterstains are used, they are different colour from the primary stain
- applied after the primary stain so that cells that did not retain the primary stain take on the colour of safranin
- colouring the bacteria that was destained
- bacteria that have retained the crystal violet even after destained will be stained with safranin, but since crystal violet is much darker the cells remain purple
crystal violet basic stain
cv particles get trapped in the peptidoglycan layer of the cell wall
- gram positive bacteria have a thick layer of peptidoglycan, therefore many dye particles are trapped
- in gram negative bacteria a semi permeable outer membrane only allows a few dye particles to pass through and get tapped in the thin peptidoglycan layer but the negative charge on the outer surface of the cell also attracts the crystal violet
iodine a mordant
mordant can be anything that acts as a helper to increase the intensity of the primary stain
heat is used as a mordant for some stains
for gram stain iodine is the mordant
iodine acts by forming complexes with the crystal violet particles, causing them to increase in size and be trapped more effectively by the peptidoglycan
heat fixing
attaches the cells to the slide so that they dont come off after repeated washes
KOH string test
-rapid means of confirming the gram reaction
-by adding 3% KOH to bacteria , two reactions may happen
-gram positives will exhibit no reaction at all
-gram negative will form a slimy viscous solution that strings as the loop is pulled away from the slide
the gram negative cell walls are disrupted by the alkaline conditions created by the KOH and the strings form from the release of DNA from the inside the cell
gram positive cell walls are not susceptible to these conditions and dont break down as readily
m. lu
umbonate elevation yellow circular entire margin opaque glossy
