Lecture 3 -- General Principles of Diagnostic Microbiology Flashcards
Microorganisms in nature exist as _______________________
mixed cultures - difficult to study
In order to characterize microorganisms, one must :
isolate the different species from a specimen into a pure culture → Helps to see its molecular fingerprint
What is a culture media ?
Nutrient material used to grow + isolate microorganisms, type depends on …
- Source of sample tested
- Species suspected to be in sample
- Nutritional requirement of the suspected organisms
What is an inoculum ?
Small portion of sample that is used to inoculate different media to isolate microorganisms
Isolated colony = When 1 bacterial cell hits agar & grows / divides to make between 1 mill - 1 bill identical copies (becomes visible colony)
Methods of Inoculation
- Streak plate method
- Spread plate method
- Pour plate method
Streak plate method
Most common used
GOAL –> Isolate + purify specific bacteria species from a mixed sample
blood sample taken, streaked along quadrant along media / food on petri dish
[ see pic ]
- Sterilize
- Streak starting from new spot (Touches the previous spot)
- Throw out, sterilize do next quadrant
- Repeat
- Put in incubator
- See if u had growth or isolated colonies
Spread plate method
Take KNOWN VOLUME of blood sample , make dilutions in saline + add a bit of that → Spread by hockey stick on agar medium
- 1 dilution gives countable numbers of colonies
- As dilution goes up → You get countable number
GOAL –> Enumerate / quantify how much bacteria in sample
Pour plate method
Not as often used for isolating BUT was 1st technique invented
Bacteria grows inside agar, used for enumeration of bacteria
- Mix KNOWN VOLUME of blood sample with agar / food → Swirl around, pour on plate then put in incubator
- Food is liquid but has agar to solidify
- Challenge was to have it hot enough in test tube so that the food stayed liquid, but if it was too hot it could kill whatever is in your sample
- Some colonies may be embedded inside agar (need to go digging – challenging)
Incubation
Inoculated media must then be incubated at the appropriate temperature (normally 37ºC) to allow microorganisms to grow and multiply
When enough cells have divided (~a few million), colonies are formed which are visible to the naked eye
Preservation of Pure Cultures
Short-term preservation : Cultures can be stored in medium refrigeration temps (4 - 10ºC)
Long term preservation : Requires cultures to be maintained using one of the following approaches …
- Frozen in liquid nitrogen (-196ºC)
- Frozen in special freezer (-70ºC - -120ºC)
- Lyophilization (freeze drying) – Dehydration followed by vacuum sealing (most stable form of storage)
2 approaches to the study of microorganisms after being isolated as a pure culture :
- Colonial morphology (“image” of colony as a whole → form, elevation, margins)
-
Cellular morphology (suspension of individual cells, requires the use of microscope)
- Size of most microbial cells and viruses is in range of nanometer - micrometer so there is a need for magnification
- A microscope’s useful magnification is limited by its resolving power – (better resolving, more expensive microscope so you can see things apart as separate
What is resolution ?
Ability to distinguish 2 closely located objects as separate, distinct entities
- Fixed by the wavelength of light used & by the optical properties of the lenses
Light vs Electron Microscopy
Light microscope – System of lenses used to manipulate the path a light beam travels between the specimen & the eye
Electron Microscopy – The short wavelength of the electron beam by system of magnetic fields as compared to light allows for greater resolving power (0.003 um)
- Magnification of 1 million x is possible
- Transmission EM – Stain whole or thin sections of specimen with heavy metals
- Scanning EM – Electron beam moves back / forth to generate 3D image of cell surface
Importance of studying detailed morphology of microorganisms :
- Absence or presence & characteristics of cellular structures help classification
- Morphology of cells help them to respond to environment (eg. Extracellular structures such as capsules can make microorganism more pathogenic)
Basic Protocol for Staining of Microorganisms
- A thin film of specimen (smear) is placed onto a clean microscope slide and air dried
- The dried smear is fixed by heat to make microorganisms stick to glass slide – Glass slide passed over bunsen burner / flame 3-4 times
- Will denature some proteins – Allows whatever smeared to stick to slide, making sure everything stays there & doesnt mess up shape
- If u overheat & denature everything, just blob of dye - Stain with 1 or more dyes prior to viewing with microscope
Simple Staining vs Differential Staining
Simple staining : Staining by a single general dye, all microorganisms in a specimen are the same colour
- Allows the observation of size, shape, number + arrangement of cells
- eg. Methylene Blue staining
Differential staining : 2 or more special dyes are used to observe differences between microbial cells or parts of cells
- eg. Acid fast stain – Carbolfuchsin & methylene blue used to differentiate acid-fast bacteria such as mycobacterium from other non-acid-fast bacteria
- eg. Gram stain – Characterizes bacteria into Gram positive or Gram negative (ANTIBIOTICS)
Difference in staining is determined by __________________________________
different cell wall structures
Gram Staining
Gram stain has long been considered to be one of the most important staining procedures in microbiology!
- Hans Christian Gram
- Has to do with the cell wall of a bacterium …
What are characteristics of the *cell wall ?
Rigid structure giving characteristic shape of bacterial cell
- Essential for cell growth + division
- Gram-negative cell wall usually thinner than Gram-positive cell wall
- In eubacteria, shape determining part is rigid material called peptidoglycan; archaebacteria do NOT have peptidoglycan
Gram-positive cell wall
Thick structure made up mainly of thick layer of peptidoglycan - Ethanol added to this will cause part to wash off & part to remain, which is why it remains purple
- Pink will bind to cell wall, but purple masks pink bc there is a bit of peptidoglycan left
- Teichoic acids often found attached to peptidoglycan to give negative charge to help transport of positive ions and storage of phosphorus
Gram-negative cell wall
- More complex
- Has an outer membrane covering a thin layer of peptidoglycan ( which crystal & iodine bind to )
- Outer membrane anchored to peptidoglycan by a lipoprotein, & contains lipopolysaccharide (LPS)
- Selective barrier based on size and charge of molecules
- Alcohol increases permeability of Gram-negative outer membrane but shrinks pores of Gram positive peptidoglycan
ONLY Gram Neg have endotoxins !!! (liberated when cell wall is destroyed) bc gram pos do not have second / outer wall
Gram-Staining Steps
- Flood slide with crystal (or gentian) violet, wash with running tap water
- Flood with Gram’s iodine, wash with water
-
MOST CRITICAL & MOST AFFECTED BY TECHNICAL VARIATIONS IN TIMING + REAGENTS → Carefully decolorize with 95 % ethanol (wash with water)
- Everything looks purple at first, but gram neg becomes invisible (need to stain it again to be able to observe what is actually there) - Flood with safranin (pink color), wash with water, then airdry or blot with absorbent paper
Gram pos remains purple even after pink dye, while gram neg becomes pink !
Other Types of Staining
- Endospore staining – Malachite green applied with heat to penetrate spores followed by counter-staining with safranin
- Capsule staining – Treat with copper sulfate before staining to visualize capsule as a clear zone surroundings cells
- Flagella staining – Use of mordant to thicken flagella before staining to visualize
Fluorence Microscopy
Helps reveal objects of interest in black background
- Special dye which fluoresces at a specific wavelength is visualized using a light microscope equipped with the appropriate filters
Immunofluorescence – Antibodies tagged with dyes, common + important aspect of fluorescence microscopy
- In an immunofluorescence assay, make antibodies visible as they bind to any part of the microorganism