Lecture 3 -- General Principles of Diagnostic Microbiology

Question 1

Microorganisms in nature exist as _______________________

Answer 1

mixed cultures - difficult to study

Question 2

In order to characterize microorganisms, one must :

Answer 2

isolate the different species from a specimen into a pure culture → Helps to see its molecular fingerprint

Question 3

What is a culture media ?

Answer 3

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

Question 4

What is an inoculum ?

Answer 4

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)

Question 5

Methods of Inoculation

Answer 5

• Streak plate method
• Spread plate method
• Pour plate method

Question 6

Streak plate method

Answer 6

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

Question 7

Spread plate method

Answer 7

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

Question 8

Pour plate method

Answer 8

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)

Question 9

Incubation

Answer 9

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

Question 10

Preservation of Pure Cultures

Answer 10

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)

Question 11

2 approaches to the study of microorganisms after being isolated as a pure culture :

Answer 11

1. Colonial morphology (“image” of colony as a whole → form, elevation, margins)
2. 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

Question 12

What is resolution ?

Answer 12

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

Question 13

Light vs Electron Microscopy

Answer 13

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

Question 14

Importance of studying detailed morphology of microorganisms :

Answer 14

• 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)

Question 15

Basic Protocol for Staining of Microorganisms

Answer 15

1. A thin film of specimen (smear) is placed onto a clean microscope slide and air dried
2. 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
3. Stain with 1 or more dyes prior to viewing with microscope

Question 16

Simple Staining vs Differential Staining

Answer 16

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)

Question 17

Difference in staining is determined by __________________________________

Answer 17

different cell wall structures

Question 18

Gram Staining

Answer 18

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 …

Question 19

What are characteristics of the *cell wall ?

Answer 19

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

Question 20

Gram-positive cell wall

Answer 20

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

Question 21

Gram-negative cell wall

Answer 21

• 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

Question 22

Gram-Staining Steps

Answer 22

1. Flood slide with crystal (or gentian) violet, wash with running tap water
2. Flood with Gram’s iodine, wash with water
3. 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)
4. 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 !

Question 23

Other Types of Staining

Answer 23

• 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

Question 24

Fluorence Microscopy

Answer 24

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

Question 25

**Bacteria**

Answer 25

- Small (0.75 - 1.25 um in diameter/width) but have a much higher ratio of SA:V than larger, more complex organisms - Higher metabolism - Faster growth - Replication rate of 18 - 20 mins

Question 26

All bacteria have 1 of 3 ***basic shapes***

Answer 26

- *Spherical cells* (called ***coccus***) – Usu round, sometimes ovoid - *Cylindrical* or *rod shape* (called ***bacillus***) – Vary in width and length, ends can be square, rounded, tapered or pointed - *Spiral* or *helical shape* (***spirillum***) - Corkscrew like shape Not all bacteria have exactly these 3 general shapes ; A few can change cell shape as it grows → ***Pleomorphic organisms***

Question 27

***Specific patterns of individual bacterial cells***

Answer 27

**Gross morphology** – Distinguishment of bacterial cells by size, shape & arrangement - *Spiral-shaped* & *rod-shaped* bacteria usually exists as **single cells** - When coccus divide in one single plane = ***diplococcus*** (pairs) [ eg. Neisseria gonorrhea ] - When coccus divide in one plane but remain attached to form chain = ***Streptococcal arrangement*** - When coccus divide at right angle to the first plane of division = ***Tetrads*** [ eg. Pediococcus ] - Further division of coccus in third plane = ***Cubical packet of 8 cells called sarcinae*** [ eg. Sarcina spp ] - Division of coccus in 3 planes in an irregular pattern = ***Grapelike clusters*** [ eg. Staphylococcus ] Not all bacteria are found in their perfect arrangement under microscope. It is useful to look for predominant patterns of arrangement

Question 28

*Specific Media Required for Isolation of a Microorganism*

Answer 28

***Chemically defined media*** – Exact composition of media know (sugars, proteins, amino acids, etc) thus can alter individual component separately ***Undefined media*** – Natural products (eg., blood, beef extract, peptone, etc.) added to media for routine lab cultivation, some components can’t be controlled - eg. blood → cannot control exact RBC amount ***When solid support needed***, 1.5 % agar is used ***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 ***Media for growing yeasts*** – All fungi and *heterotrophs* (requires organic substrates to get its carbon source for growth & development) Generally have higher sugar content + lower pH than bacterial media ***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 ***Selective media*** – Media designed to enhance growth of one kind of microorganism or suppress growth of other kind of microorganism or both - Most of agar media used will be selected for either g pos or g neg - eg. Brilliant green agar is used to isolate Salmonella species from food samples. That is, the brilliant green agar inhibits Gram-positive bacteria - eg. Phenylethanol agar that inhibits Gram negative bacteria ***Differential media*** – Used to differentiate organisms based on their unusual nutritional requirements & phenotypic haracteristic appearance in media (eg. Lysis of red blood cells, hemolytic vs. nonhemolytic bacteria) ***Selective/Differential media*** – Useful in public health / clinical microbiology - eg. *MacConkey medium agar* (bile salt and crystal violet inhibits Gram-positive bacteria thus allowing Gram-negative organisms) ***Enrichment media*** – Used to increase number of specific organism in a sample by favoring the growth of the interested species ***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

Question 29

***4 Physical Conditions Essential for Successful Cultivation of Microorganisms***

Answer 29

- Temperature - Gaseous Atmosphere - pH - Other conditions

Question 30

*Temperature*

Answer 30

Microorganisms in general can grow over a wider temperature range than more complex organisms ***Optimum growth temperature*** – Temperature at which a species grows most rapidly **Cardinal temperatures of a species of microorganisms :** Change depending on nutritional content of growth medium & are usually closer to max temp since enzyme activity increases with temp until upper limit at which enzymes is degraded is reached - *Minimum temperature* - *Optimum temperature* - *Maximum temperature* ***Psychrophiles*** – Grow best from 15 - 20ºC (may die if exposed to room temperature for short time probably due to damage to cytoplasmic membrane) - Bacteria & fungi from this group found in colder waters + soils such as oceans, the Polar Regions - Can grow / survive in refrigeration temps - Particularly problematic in food spoilage (eg. psychrophilic bacteria are Pseudomonas and Flavobacterium) ***Mesophiles*** – Majority of microorganisms belong to this group. Grow best between 25 - 40ºC, but optimal is 37 (human body temp) - Saprophytic organisms grow at lower part of the mesophilic temp range - Parasitic organisms of humans and animals grow at upper part of this range (~37ºC) ***Thermophiles*** – Can grow from 40 - 85ºC but grow best between 50 - 60ºC - Mostly prokaryotes (eukaryotes cannot grow above 60ºC) - Commonly found in volcanic areas, compost heaps & hot springs - eg. Bacillus stearothermophilus - Enzymes are rapidly produced to replace those damaged by high temperatures

Question 31

*Gaseous Atmosphere*

Answer 31

Right combination of gases such as oxygen, carbon dioxide, nitrogen and methane resembling that found in natural habitat essential for cultivation of microbes in vitro ***Aerobic Microorganisms :*** - Includes microbes which can grow in standard atmosphere of 21% oxygen (eg. Mycobacterium, Legionella, filamentous molds) - Require more energy than those microorganisms & no problem with oxygen supply when grown on surface of plates, require higher levels of carbon dioxide in broth (eg. Neisseria gonorrhoeae → 5 - 10% carbon dioxide) - Can use candle jar or more complex gas jars *Bacteria come to TOP of test tube to receive more oxygen* ***Anaerobic Microorganisms :*** May be poisoned by oxygen, cannot grow in air atmosphere, does not use oxygen for energy-yielding chemical reactions - Wide range in oxygen tolerance, seen with Clostridium perfringens, Clostridium tetani and Methanobacterium. Toxicity of oxygen due to production of superoxide radical, hydrogen peroxide and hydroxyl radicals - Aerobes protect against these radicals by producing enzymes such as superoxide dismutase, catalase and peroxidase - Anaerobic jars, anaerobic chamber or anaerobic glove box can be used cultivate anaerobes *Will avoid oxygen, BOTTOM → Sensitive to oxygen radicals* ***Facultative Microorganisms*** – Grow in air atmosphere but can also grow anaerobically - Do not need oxygen but can use it for chemical reactions - Prefer oxygen but can still otherwise grow / survive eg. E. coli and Saccharomyces cerevisiae (common baker’s yeasts) *Near top + a little scattered bottom* ***Aerotolerant Anaerobes*** – Prefer anaerobic conditions, but some oxygen is OK (all over) ***Microaerophilic Microorganisms*** – Can use oxygen for chemical reactions, but require a VERY SPECIFIC CONCENTRATION (will not grow if too much / little) - Grow best between 1 to 15% oxygen level (eg. Campylobacer jejuni) *Concentrate around specific portion of test tube*

Question 32

*pH*

Answer 32

- Regardless of the external pH, microorganisms **must maintain** *intracellular pH at ~7.5* (this is done by the ability of the cell to expell or uptake hydrogen ions) - Most bacteria can grow between pH 4 - pH 9 (optimum pH normally between 6 - 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 ]

Question 33

*Other conditions*

Answer 33

*Water & sometimes light* **Osmotic pressure :** - In a *hypertonic solution*, there is a higher solute concentration in environment than cell cytoplasm, so the cell loses water and eventually shrivel up - In a *hypotonic solution*, lower concentration of solutes in the environment leads to inflow of water resulting cell rupture - In an *isotonic solution*, no net flow of water occurs, resulting in normal cell growth

Question 34

***Direct vs Indirect ELISA***

Answer 34

**Direct :** Detects your antigen (antigen unique to pathogen) - If there is colour = positive (how much colour gives idea how much antigen you had) - eg. Pregnancy tests detect specific hormone – acts as the antigen, heavy metals **Indirect :** Detects if patient has made Ab (looking for patients immune host response) / if patient has made Ab against HIV - Well coated with HIV (not Ab) → pass all Ab franco has made - They will bind if Ab bind to HIV, if they don’t → get washed away if there is a lot of colour → a lot of Ab has been made