3 Flashcards
Nutrient material called ____ ______ used to grow and isolate microorganisms
culture media
what factors are considered when picking a media when trying to determine a bacteria
i. Source of sample tested
ii. Species suspected to be in sample
iii. Nutritional requirement of the suspected organisms
Methods of inoculation include
i. Streak plate method
ii. Spread plate method
iii. Pour plate method
what temp is inncoulated media kept at
37 degree celsius
cell colonys
Each colony is derived from a single cell and all cells within a colony are
identical to each other. Different species have different colonial morphologies therefore can be used as mean
of distinguishing different species in a mix culture
Longterm preservation requires cultures to be maintained using one of the following approaches:
frozen in liquid nitrogen (-196ºC)
ii. Frozen in special freezer (-70ºC to -120ºC)
iii. Lyophilization (freeze drying) - dehydration followed by vacuum sealing (most stable form of
storage)
Two approaches to the study of microorganisms after being isolated as a pure culture
colonial morphology and the cellular morphology. The latter requires the use of microscope.
size of micribials
range of nanometer to micrometer and so there is a need for magnification by using a microscope
what is resolution
ability to distinguish two closely located objects as separate, distinct entities
-Resolution is
fixed by the wavelength of light used and by the optical properties of the lenses.
what is a Light microscope
system of lenses used to manipulate the path a light beam travels between the specimen
and the eye
what is an Electron microscope
a beam of electrons controlled by a system of magnetic fields used in place of the light source of a light microscope.
basic protocol for staining of microorganisms
a. A thin film of specimen (smear) is placed onto a clean microscope slide and air dried;
b. The dried smear is fixed by heat to make microorganisms stick to glass slide;
c. Stain with one or more dyes prior to viewing with microscope.
Simple staining
staining by a single general dye colours all microorganisms in a specimen and allows
the observation of size, shape, number and arrangement of cells.
-e.g. methylene Blue staining
Differential staining
-two or more special dyes are used to observe differences between microbial cells or parts of cells
-acid fast stain
- carbolfuchsin and methylene blue used to differentiate acid-fast bacteria such
as Mycobacterium from other non-acid-fast bacteria
- Gram stain - characterize bacteria into two groups: Gram positive and Gram negative.
Gram stain has to do with the cell wall of a bacterium.
Gram stain has to do with the cell wall of a bacterium.
cell wall
- rigid structure giving characteristic shape of bacterial cell;
- essential for cell growth and division;
- Gram-negative cell wall usually thinner than Gram-positive cell wall;
- in eubacteria shape determining part is rigid material called peptidoglycan;
- archaeobacteria do not have peptidoglycan
Gram-positive cell wall
- thick structure made up mainly of thick layer of peptidoglycan;
- teichoic acids often found attached to peptidoglycan to give negative charge to help transport of positive
ions and storage of phosphorus
(ii) Gram-negative cell wall
- has an outer membrane covering a thin layer of peptidoglycan (i.e., in periplasmic space between outer and
cytoplasmic membranes); - outer membrane anchored to peptidoglycan by a lipoprotein;
- another structure found only in outer membrane is lipopolysaccharide (LPS);
- outer membrane is a selective barrier based on size and charge of molecules;
- in Gram strain, alcohol increases permeability of Gram-negative outer membrane but shrinks pores of Grampositive peptidoglycan.
when is Fluorescence Microscopy a useful tool
Fluorescence Microscopy is a useful tool when trying to reveal only objects that are of interest in an otherwise
black background
using light mictoscope
-e.g a fluorescent dye is attached to a specific antibody; Any attachment of the labelled antibody to microorganisms in the specimen will fluoresce under the appropriate conditions and can be easily detected
Electron Microscopy
short wavelength of the electron beam as compared to light allows for greater resolving power
- Transmission electron microscopy requires staining with heavy metals of whole specimen or
slicing of microorganisms into thin section
- scanning electron microscopy, an electron beam moving back and forth generates a 3-dimensional image of cell surface of microorganisms coated with a fine
film of metal.
*Importance of studying detailed morphology of microorganisms include
a. absence or presence and characteristics of cellular structures help classification
b. morphology of cells help them to respond to environment.
(e.g., extracellular structures such as capsules can make microorganism more pathogenic)
why are bacteria mostly used in biology studies
Bacteria are small (0.75 to 1.25 um in diameter/width) but have a much higher ratio of surface area to volume
than larger, more complex organisms
-high rate of metabolism, growth and replication
what are the 3 shapes of bacteria?
Spherical cells (called coccus) - usually round, sometimes ovoid
b. Cylindrical or rod shape (called bacillus) - vary in width and length, ends can be
square, rounded, tapered or pointed.
c. Spiral or helical shape (spirillum) - corkscrew like shape
what are bacterias that change shapes as they grow called
pleiomoric organisms
what patterns do bacterial cell have
a. spiral-shaped and rod-shaped bacteria usually exists as single cells
b. when coccus divide in one plane = diplococcus (pairs) e.g., Neisseria gonorrheae
c. when coccus divide in one plane but remain attached to form chain = streptococcal arrangement,
e.g., Streptococcus spp.
d. when coccus divide at right angle to the first plane of division = tetrads, e.g., Pediococcus
e. a further division in third plane = cubical packet of 8 cells called sarcinae, e.g., Sarcina spp.
f. division in 3 planes in an irregular pattern = grapelike clusters, e.g., Staphylococcus
Size, shape and arrangement of bacterial cells is referred to as?
gross morphology.
In order to isolate a microorganism, specific media is required:
1- Chemically defined media. - Exact composition of media know thus can alter individual component
separately.
Undefined media - natural products (e.g., blood, beef extract, peptone, etc.) added to media for routine lab
cultivation.
2- When solid support needed, 1.5 % agar is used.
3- Media for growing bacteria - requirements varies between different bacteria.
Microorganisms with demanding nutritional requirements are known as fastidious. They require complex,
undefined media for cultivation.
4- Media for growing yeasts - all fungi and heterotrophs (requires organic substrates to get its carbon source
for growth and development).
- generally have higher sugar content and lower pH than bacterial media.
5- Media for anaerobes:
-anaerobes are those that tolerate little or no oxygen (sometimes grown deep down in agar or put media in tall test tubes.
6- Selective media - media designed to enhance growth of one kind of microorganism or suppress growth of
other kind of microorganism or both.
For example, brilliant green agar is used to isolate Salmonella species from food samples. That is, the brilliant
green agar inhibits Gram-positive bacteria. Another example is phenylethanol agar that inhibits Gramnegative bacteria.
7- Differential media - used to differentiate organisms based on their unusual nutritional requirements and
characteristic appearance in media (e.g., lysis of red blood cells, hemolytic vs. nonhemolytic bacteria).
8- Selective/Differential media - useful in public health microbiology - e.g., MacConkey medium
(bile salt and crystal violet inhibits Gram-positive bacteria thus allowing Gram-negative organisms).
9- Enrichment media - used to increase number of specific organism in a sample by favoring the growth of
the interested species.
10-Tissue culture media - plant or animal cells grown in lab in specialized media used to cultivate
viruses in vitro since viruses can only replicate inside living host cells.
temperature
- Microorganisms in general can grow over a wider temperature range than more complex organisms (e.g.,
Bacillus subitlis can grow from 8 to 53ºC). - Temperature at which a species grows most rapidly is the optimum growth temperature.
- Cardinal temperatures of a species of microorganisms
40
(i) Minimum temperature
(ii) Optimum temperature
(iii) Maximum temperature - Cardinal temperatures changes depending on nutritional content of growth medium.
- Optimum temperature usually closer to maximum temperature since enzyme activity increases with
temperature until upper limit at which enzymes is degraded is reached.
Microorganisms are divided into 3 major groups based on their optimum growth temperature:
Psychrophiles - grow best from 15 to 20ºC (may die if exposed to room temperature for short time
probably due to damage to cytoplasmic membrane). Bacteria and fungi from this group found in
colder waters and soils such as oceans and the Polar Regions. Particularly problem of food
spoilage, e.g., psychrophilic bacteria are Pseudomonas and Flavobacterium.
Mesophiles - majority of microorganisms belong to this group. Grow best between 25 to 40ºC.
Saprophytic organisms grow at lower part of the mesophilic temperature range.
Parasitic organisms of humans and animals grow at upper part of this range (~37ºC).
Thermophiles - can grow from 40 to 85ºC but grow best between 50 to 60ºC. Mostly prokaryotes
(eucaryotes cannot grow above 60ºC). Commonly found in volcanic areas, compost heaps and
hot springs. E.g., Bacillus stearothermophilus. Enzymes are rapidly produced to replace those
damaged by high temperatures
Gaseous Atmosphere
- Aerobic Microorganisms
-21% oxygen. E.g., Mycobacterium, Legionella, filamentous molds
- Require more energy
2.Anaerobic Microorganisms
- may be poisoned by oxygen
- Wide range in oxygen tolerance
-Anaerobic jars, anaerobic chamber or anaerobic glove box can be used cultivate
anaerobes - Facultative Microorganisms
-grow in air atmosphere but can also grow anaerobically
-cna use oxygen for chemical reaction
-e.g. e. coli - Microaerophilic Microorganisms
-use oxygen for chemical reaction
-1 to 15% oxygen level.
- E.g., Campylobacer jejuni.
pH
-optimal diff for various organism
-microorganisms must maintain intracellular pH at ~7.5 (this is done by the ability of the cell to expel or uptake hydrogen ions.
-minimum pH of 4 and maximum pH of 9. Optimum pH normally lies between 6
and 8.
-molds and yeasts generally have a broader pH range for growth than bacteria. Optimum pH is ~ 5 to 6.
- growing cells release acidic or alkaline waste products into the growth medium environment. Without
buffering of the medium, the shift can eventually inhibit growth
Other conditions
-water, and light
- osmotic pressure (hypertonic, hypotonic, isotonic)
