Lecture 3- Diagnostic Microbiology Flashcards
Isolation of pure culture from specimen
In order to characterize microorganisms, one must isolated different species from specimen
Culture Media
Used to isolate microorganisms
Media type depends on:
- Source of sample tested
- Species suspected to be in sample
- Nutritional requirement of suspected organisms
Inoculum
- Small small portion of the sample
- Used to inoculate different media to isolate microorganisms
Methods of Inoculation
- Streak plate method
- Spread plate method
- Pour plate method
After inoculation you must..
Incubate at 37C (allows organism to multiply and divide)
Colonies
- A few million divided cells, visible to the naked eye
- Each cell within the colony is the same
- Colonial morphologies can be used as a mean of distinguishing
Short Term Preservation of Pure Cultures
- Stored in medium refrigeration temperature
- 4 - 10 C
Long Term Preservation of Pure Cultures
- Frozen in liquid nitrogen (-196C)
- Frozen in special freezer (-70 to -120C)
- Lyophilization (freeze drying), most stable form for storage.
Identification of Microorganism After Obtaining Pure Culture
- Colonial morphology
(Form, Elevation and Margin) - Cellular morphology (needs microscope)
Resolution
- The ability to distinguish two closely located objects as separate distinct entities
- fixed by the wavelength of light use and by the optical properties of the lenses
Light Microscope
System if lenses used to manipulate the path light beam travels between the specimen and the eye
Electron Microscope
- A beam of electrons controlled by a system of magnetic fields used in place of the light source of a light microscope
- TEM –> stain with heavy metals
- SEM –> 3D image
Streak Plate
- Comes directly from specimen
- Isolates the colonies
- From one streak to another
- MOST COMON
Pour Plate
- Place sample inside petrie dish
- Add liquid agar
- Problem: Colonies are embedded with the agar
- Used for enumeration of bacteria
Spread Plate
- Used because you don’t want to know IF, you want to know HOW MUCH
- Dilutions
- Spread all over the plate
Staining Techniques
- thin film of specimen (smear) is placed onto a clean microscope slide and let air dry
- dried smear is fixed by heat to make micro-organisms stick to the glass slide
- stain with one or more dyes prior to viewing
Simple Staining
- Staining by a single general dye colours all micro-organisms in a specimen
- Allows the observation of size, shape, number and arrangement of cells
- Methylene Blue
Differential Staining
- When two or more dyes are used to make observational difference between microbial calls or parts of cells
Differential Staining
Exampls
- Acid fast stain
- Gram stain characterize bacteria into two groups, positive and negative
The Gram Stain
1- Flood slide with crystal violet (was with running tap water)
2- Flood with Gram’s iodine (was with water)
3- Carefully decolorize with 95% ethanol (wash with water)
4- Flood with safranin (wash with water) Air Dry
Gram Positive Stain Colours
clear, purple, purple, purple, purple
Gram Negative Stain Colours
clear, purple, purple, clear, pink
Cell Wall
- Essential for cell growth and division
- Shape of bacteria related to peptioglycan layer
- Gram negative usually thinner than gram positive
Gram Positive Cell Wall
- Thick structure made of thick layer of peptidoclycan
- Teichoic acids often found attached to pep. to give negative charge to help transport positive ions and storage of phosphorus
Gram Negative Celle Wall
- More complex
- Has outer membran convering a thin layer of peptidogycan (anchored by lipoprotein)
- LPS is found in the outer layer
- Outer layer is selective based on size and charge
MORE HERE
Other Stains
- Endospore staining
- Capsule staining
- Flagella staining
MORE HERE
Fluorescence Microscopy
- Dye fluoresces at specific wave length
- Antibodies tagged with dyes are common
MORE HERE
Electron Microscopy
The short wavelength of the electron beam. Greater resolving power (0.003um)
Scanning election microscopy
Transmission electron microscopy
Importance of studying detailed morphology of micro-organisms
- absence or presence and characteristics of cellular structure help classification
- morphology of cells helps them respond to environment
Bacteria
- Small
- Higher surface area
- Higher metabolism
- Faster growth
- Replication rate (20 Minutes)
Bacterial Shapes
- Spherical cells
- Cylindrical or rod shapes
- Spiral or helical shape
Spherical cells
Round, sometimes ovid
Cylindrical or rod shapes
Called bacillus, ends can be round square, tapered or pointed
Spiral or helical shape
Sperillum - corkscrew like shape
Chemically Defined
Exact composition known
Chemically undefined
Some component cant be controlled (blood)
Agar
1.5% used to make solid growth
Enrichment media
- Increases # of specific bacteria in sample by favouring growth of interested species
Tissue Culture Media
- For cultivating viruses, derived of plant or animal cells
Media Requirements
- Bacteria, requirements vary
- Yeasts, high sugar, low pH
- Anaerobes
Selective Media
- enhance growth of one bacteria or suppression of another
Differential Media
- Differentiate bacteria based on their nutritional requirement and phenotypic characteristics
MacConkey
- Selective/Differential Media
4 Main Conditions Make up Physical Conditions Essential for Successful Cultivation of Micro-organisms
- Temperature
- Gaseous Atmosphere
- pH
- Other
Temperature
- Micro-organism can grow over a wider range than complex organisms
Optimum Growth Temperature
- Temperature at which a species grows most rapidly
Cardinal Temperature
- Minimum
- Optimal
- Maximum
- changes depending on nutritional content of growth media
Optimal Temperature
- usually closer to max temp
- at that point enzymes are degraded
3 Temperature Groups
- Psychrophiles
- Mesophiles
- Thermophiles
Psychrophiles
- grow from 15 - 20
- may die is exposed to room temp
- found in colder water and soils like oceans and polar regions
- problem of food spoilage
- Listeria
Mesophiles
- Majority of microorganisms
- 25-40
Thermophiles
- 40 - 85 C
- but grow best between 50-60
- found in volcanic areas
Oxygen requirements
- Aerobic Microorganisms
- Anaerobic Microoganisms
- Facultative Microorganism
- Microareophillic Microorganism
Aerobic Microorganisms
- Grow in standard atmospheric pressure (21% oxygen)
- Requires more energy
Anaerobic Microoganisms
- Poisoned by oxygen
- Does not use oxygen for energy
Facultative Microorganism
- Grows in air atmosphere but can also grow anaerobically
- Does not need oxygen but can use it
Microareophillic Microorganism
- Can use oxygen for chemical reactions
- Grow best between 1 and 15% oxygen level
pH
- Regardless of external pH, internal pH must remain at 7.5
- Growth observed at pH values of 4-9
Water and Light
Can be important for certain microorganisms
Osmotic Pressure
- Hypotonic
- Hypertonic
- Isotonic
Antibodies: ELISA Test
Direct and Indirect
Direct ELISA Test:
Looking for
- More colour, more antigen
- Good for looking at the antigen
- Steroid testinging
Indirect ELISA Test:
Looking For
- Have you made antibodies against something?
- more colour, more antibody
- Pregnancy test
