Analytical Techniques Flashcards
What is the difference between a monocular and a binocular?
Monocular= One eyepiece
Binocular= Two eyepiece
What is the interpupillary distance on a microscope?
The distance between the 2 eyepieces
What is the equation for total magnification?
Total magnification= eye piece × objective
What is the Kohler illumination procedure used for?
A procedure for setting up and adjusting the microscope to achieve the best possible combination of contrast and resolution.
What is the method for the Kohler Illumination procedure?
- Focus microscope on specimen
- Close field iris until Illumination
disc is smaller than the field of view - Adjust sub- stage focus control (condenser focus) until edges if the disc come into sharp focus, looking like a 20p piece.
- Use the condenser centring screws to place the lit area in the centre of the field of view.
- Open the field iris until the edges of the disc can no longer be seen
- Adjust the condenser iris to obtain best resolution/ contrast. You will need to readjust this when changing objective lens
Which objective lens should only be used with oil immersion?
100×, keep oil away from all other lenses
What is the method for oil immersion?
- Ensure your specimen is in the centre field of view, using the 100× objective lens
- Use the course focus to wind down the stage and place a small drop of oil onto the slide over the spot of light
- Use the course focus to bring the stage up until the slide is in contact with the oil and compresses the oil slightly.
- Look through the eyepiece and use the focus to very slowly move the stage away from the oil and bring your specimen into sharp focus.
What are the steps for the vernier scale?
- Put slide well seated in the slide clip and place your specimen in the centre of the field of view.
- Read the scale at the back of the stage (x axis first), look for the number on the large scale which is just below the zero of the small scale (this is the whole number)
- Now look for a line on the small scale which lines up with a line on the large scale, this gives the decimal fraction (read off the small scale).
- If both 0 and 10 on the small scale line up with the lines on the large scale the reading is a whole number
- Record the reading, now do the same using the scale on the y axis.
- Should give 2 numbers as a reference for future
Why do we need to stain bacteria?
• Visualisation
• Normally colourless
• Use dyes or stains to highlight differences
•Fast, cheap and simple
•Shows cell morphology
•Identify structures
•Two classifications- Simple and differential
What are dyes/ stains?
•Chemicals containing chromophores
•Chromophores impact colour
•Usually salts with one of the ions coloured e.g. methylene blue
*Methylene blue chloride dissociated in water into a positively charged methylene blue ion (blue colour) and negatively charged chloride ion (colourless).
What are the properties of basic dyes?
• Positive charge
• Reacts with negatively charged materials
• Bacterial cytoplasm negatively charged (slightly) in certain environments
•Attracts and binds with basic dyes
• Examples- crystal violet, safranin, basic fuchsia and methylene blue
What are the properties of acid dyes?
• Negatively charged chromatophores
• Repelled by bacterial surface and deposit around the organism
• Stain the background and leave microbe transparent.
• Examples- nigrosine and Congo red.
What are the two different types of dyes?
Basic and acid dyes
What is the method to collect a sample for a serial dilution (bacteria)?
- Select area to use swab
- Wearing gloves, place 10cm× 10cm template onto the surface
- Dip a clean swab in the sterile distilled water and swab the whole area of the template
- Repeat this until the entire surface in the template is wet (approx 10 times)
- Place the swab onto the labelled tube of sterile water and mix well
What is the method for the serial dilution (once the sample is collected) using distilled water to create dilutions of the sample?
- First label petri dishes. In the safety cabinet, using aseptic technique prepare a 1/10 dilution of your dirty cage sample by pipetting 1cm³ of your sample into a tube with 9cm³ of sterile water (ensure this is labelled appropriately).
- In the safety cabinet prepare 1/100 dilution of your dirty cage sample by pipetting 1cm³ of your 1/10 dilution into a tube with 9cm³ of sterile water.
- In the safety cabinet prepare 1/1000 dilution of your dirty cage sample by pipetting 1cm³ of your 1/100 dilution into a tube with 9cm³ of sterile water.
What is the method for plating up the agar plates to create a sample after the dilution has been made? (Serial dilution)
- In the safety cabinet using aseptic technique pipette 0.1cm³ of your dirty sample onto a petri dish with agar, use a sterile spreader to spread out the sample (take care not to cut up the agar).
- Repeat step 1 for your 1/100 dilution and 1/1000 dilution.
- Elastic band your plates together and place in the 20°C incubator for 48 hours, agar facing down.
- Place all used swabs and spreaders into an autoclave bag, place used tubes into rack for autoclaving.
- After 48hours of incubation remove and store in the fridge.
Where are bacteria found?
Found almost everywhere on Earth even where other forms of life are rare or absent
What are the general characteristics of bacteria?
• Single celled (smallest is 0.1 micrometres and largest is 6×60 micrometers)
• Diverse (vary in size, shape and habitat)
• Different types will produce different looking colonies this is termed the colony morphology and colony morphology is one of ways scientists can identify bacteria.
What are the different bacterial characteristics?
• Surface texture
•Size
•Margin
•Colony shape and grouping
•Elevation
•Opacity
•Chromogenesis
What different specific shapes can bacteria be?
•Bacili- Rod
•Cocci- Ovoid
•Spirilla- Cylindrical
What are the different types of Cocci bacteria?
•Micrococcus sp: commonly associated with the skin
•Staphylococcus sp: Also common on the skin
•Streptococcus sp: Commonly found in the mouth
What are the different types of Bacilli bacteria?
•Bacillus sp: Commonly found in soil and many food products
•Bacullus cereus: causes mild food poisoning
•Bacillus anthracis: causes anthrax in animals
What are the different types of Vibrio bacteria?
•Vitrio sp: Waterborne bacteria, causing severe disease through water and faecal contamination
•Vibrio cholerae: Causative agent of cholera
•Other Vibrio sp: mainly cause food poisoning and commonly found in raw seafood
What are the different types of spiral bacteria?
•Bacteria possessing flagella and are highly motile
•Trepnema palliclum: causes syphilis in humans
•Helicobacter heilmannii: commonly causes gastric ulcers
Can bacteria live in groupings? Give an example…
While many live alone, many group together in colonies for example, cocci….
•Diplococcus: are in sets of two (diplo= double, two)
•Streptococcus: are in chains (strepto= bent, twisted, pliable)
•Staphylococcus: are in clusters (staphylo= a bunch of grapes)
What are the different types of opacity that bacteria can be?
• Transparrent (clear)
•Opaque (not clear)
•Translucent (almost clear, like looking through frosted glass)
•Iridescent (changing colours in reflected light)
What is chromogenesis and the different pigments?
Chromogenesis is the colour production of bacteria by metabolic activities or the pigmentation of a colony.
•White
•Buff
•Red
•Purple
In microbiology what does monera include?
Bacteria and Archaea
In protozoology what does protista include?
Archaezoa, Protista and chromista
The kingdom of Monera consists of around how many unamed species (spp)?
2000
What are the two main sub kingdoms of the kingdom of monera?
- Archaebacteria (ancient)
- Eubacteria (true)
What are the three classifications of Archaebacteria?
- Methanogens
- Halophiles
3.Sulphur Reducers
Where are methanogens found?
•Found in bogs and as symbionts in herbivores where they ferment cellulose
•Some can live in water of up to 110°C
What are the properties of halophiles and where are they found?
•Live in salt water and are photosynthetic
•They can regulate osmotic pressure in concentrations of up to 36% NaCl
•Tolerance of pH up to 11.5
What are the properties of Sulphur reducers and where are they found?
•Theroacidophilic: extremophilic microorganism that is both thermophilic and acidophilic= it can grow under conditions of high temperature and low pH
•Found in sulphur springs
•Oxidises at high temperatures at pHs down to 1
What is the difference of a prokaryotic cell compared with a eukaryotic cell?
In prokaryotic cells…..
•Genetic material not in a nucleus bound by nuclear envelope
•Organelles such as mitochondria, chloroplasts, golgi apparatus, endoplasmic reticulum and lysosomes are all absent
What are the 6 major classes in bacterial classification?
- Eubacteria
2.Myxobacteria
3.Actinomycetes
4.Spirochaetae
5.Mycoplasmas
6.Rickettsiae
What are the characteristics of Eubacteria (distribution, role…)?
•The largest class often called ‘true bacteria’
•Includes some of ‘the most common infectious agents (Escherichia/ E.coli, Streptococcus, Staphylococcus)
•Distribution (soil, water and parasites)
•Ecological role (decomposers, symbionts, pathogens)
•Diseases e.g. tetanus, diptheria
What are the characteristics of Myxobacteria (distribution, role…)?
•Slime producing soil bacteria with complex life cycles including spare production (sporulation) e.g. Myxococcus, cytophaga
•Distribution (soil, some aquatic areas)
•Ecological role- decomposers
•No diseases
What are the characteristics of Actinomycetes (distribution, role…)?
•Filamentous forms are able to cause infectious diseases but also make antibiotics to cure them (actinomyces, lactobacillus)
•Distribution- soil, parasites
•Ecological role- decomposers, pathogens, some produce antibiotics
•Diseases- Tuberculosis, leprosy
What are the characteristics of Spirochaetae (distribution, role…)?
•Largely pathogens, they are morphologically distinct, with extremely long corkscrew spiral shapes (spirochaeta, leptospira)
•Distribution- Parasites and aquatic (polluted)
•Diseases e.g. Treponema causing syphilis
What are the characteristics of Mycoplasmas (distribution, role…)?
•Intracellular parasites and the smallest of all known prokaryotes
•Contain half of DNA of larger bacteria (250 genes)
•Lack cell walls
•Distribution- Intracellular parasites
•Colonise respiratory and urogenital tracts of mammals and birds
•Resistant to penicillin
What does Rickettsiae cause and where does it live?
•Live in arthropods (lice, ticks and fleas) which are unharmed and mammals and birds which become infected (chlomidia)
•Also cause typhus
What are the different energy sources/ modes of nutrition for different types of bacteria?
•Heterotrophs- rely on performed organic material synthesised
•Autotrophs- Able to synthesise all the organic molecules needed from inorganic sources, carbon only needed in the form CO2
•Some “photosynthetic autotrophs” derive energy from light using pigments similar to chlorophyll of green plants
•Others are “chemosynthetic autotrophs” deriving their energy from inorganic chemical sources (e.g. N,S and Fe in).
What are the main tryes of heterotrophs and what do they do?
•Parasites- live on living organic matter or organisms
•Saprophytes- live on decaying organic matter
•Symbionts- to mutual benefit
•Commensals- 1 sided symbionts
What types of bacteria can/ can’t live in certain oxygen levels?
•Anaerobic bacteria- Can prosper in environments lacking free oxygen (unlike animals and most plants)
•Obligate anaerobes: Cant live with oxygen
•Methane producing bacteria (methanogenic): live in the lower gut or in swamps. Carbohydrate is fermented to lactic acid, ethanol, acetic, propianic or butyric acid.
•Aerobic bacteria tolerate or require oxygen
•Facultative anaerobes- can live with/ without oxygen
What temperature conditions are suitable for bacteria?
•Majority live and reproduce from 0-75°C
•Each has an optimum temperature for growth, those in Mammals are best suited to 35-40°C
•Psychrophiles/ cold loving bacteria can grow between 10-20°C whereas hyperthermophiles are adapted to very hot conditions (optimum >75°C)
What pH conditions are suitable for bacteria?
•Most bacteria grow best at 6.5- 7.5
•Acidophiles- 1- 5.5
•Neutrophiles- 5.5- 8
•Alkalophiles- 8.5- 11.5
How is bacteria beneficial for life on earth?
•Life on earth and the functioning of ecosystems depends on bacteria, they are the main organisms of decay
•Organic compounds are broken down from proteins 》amino acids 》ammonia (NH3)
•Bacteria play a vital role in Nitrogen fixation (atmospheric N2 to ammonia NH3)
•The earth’s atmosphere contains 80% Nitrogen (N2) but plants can’t use this so bacteria is essential to convert this to usable compounds (ammonia)
•Nodules on roots of legumes have bacteria that fix N2 as a result of energy derived from carbohydrate of their host
•Most bacteria are helpful lactobacillus and Streptococcus lactii ferment lactose to lactic acid
•Actomycetes and streptomycetes secrete antibiotics (streptomycin, terramycin, aureomycin, neomycin and erthromycin)
•There are more bacteria in the body than human cells:
~ They aid digestion
~Produce needed vitamins
~Destroy harmful organisms
When causes a disease to occur?
If bacteria produce toxins or infection spreads due to bacteria multiplying (despite our immune response) disease ensues
What is a bacteria causing disease known as?
A pathogen
What are exotoxins?
Toxins that are made from protein. They are produced by gram positive bacteria and are secreted into the surrounding environment or host tissue.
What are endotoxins made from?
Lipids and carbohydrates
What are endotoxins?
•They are associated with the outer membrane of gram negative bacteria (E. Coli)
•Endotoxins are not released until the bacteria dies
•Cause fever, body aches and weakness, damage to the circulatory system, cause disease by damaging and destroying body tissue (Streptococcus spp.)
This leads to a greater risk of invasion
What is the quantitative method for colony counting bacteria?
•Use the colony counters to count the number of colonies for each dilution on each agar plate and record this.
•The area swabbed was 100cm³, this was diluted by placing the swab into 10cm³ of sterile distilled water
•Therefore 1cm³ of solution contains the bacteria from an area of 10cm²
•Therefore the 0.1cm³of solution used to inoculate your plate contains the bacteria from 1cm²
What does the streak plate method of isolation allow?
Allows microbiologists to obtain a pure culture
Why is a pure culture required?
A pure culture is required before tests to identify bacteria can be done
What is the purpose of the streak plate method of isolation?
•Bacteria from a mixed culture are streaked over the agar surface in a pattern that deposits them further and further apart
•Towards the end of the streaking process the result should separate colonies
•Individual colonies can then be used, picked off and usually mixed with some sterile distilled water to start pure colonies.
What is the methodology for the streak plate method of isolation?
- Choose one colony and make a bacterial soup by using a sterile heated wire loop and removing the colony from the plate and putting it into 5ml of sterile water. This must be done aseptically and therefore carried out in the microbiological safety cabinet.
2.Draw a small square onto the back of your agar plate. This is where you will start your streak
- Using a swab, fill this square with your bacterial soup
- Complete your streak
What is the method of the staining technique which enables the division of two distinct groups of bacteria (gram negative and gram positive)?
This is most important differential staining technique in bacteriology
- Bacterial cells are dried on a glass slide, stained with crystal violet, then washed briefly in water.
- Iodine solution is added so that the iodine forms a complex with crystal violet in the cells
- Alcohol or acetone is added to make the crystal violet- iodine complex soluble
- The cells are counterstained with safranin, then rinsed and dried for microscopy
How does the staining technique separate bacteria into two types?
•Gram positive cells retain the crystal/ violet- iodine complex and appear purple
• Gram negative cells are decolourised by the alcohol or acetone treatment, but are then stained with safranin so they appear pink- due to differences in cell wall structure.
What properties of Gram negative and positive bacteria separate them into two types when stained?
•Gram negative bacteria have a thick wall composed of many layers of the polymer peptidoglycan.
•The thickness of this wall blocks the escape of the crystal violet- iodine complex when the cells are washed with alcohol or acetone.
•Gram negative bacteria have a thin layer of peptidoglycan surrounded by a thin outer membrane composed of lipopolysaccharide (LPS).
•The crystal violet- iodine complex is easily lost through the LPS and thin peptidoglycan layer when cells are treated with acetone. But will then be stained by the safranin afterwards.
What are examples of Gram negative and positive bacteria?
Negative:
•Eschericha coli
•Salmonella
Positive:
•Listeria
•Staphylococcus
•Streptococcus
What is required in a suitable environment to promote growth?
•Water
•Essential nutrients
•Correct gaseous environment
•Correct pH
•Correct temperature
What is a culture medium?
•Provide essential nutrients that bacteria (grown on the media or within) require in order to reproduce
What are the two types of medium?
•Nutrient broths/ liquids
•Agar plates
What temperature should unused agar plates be kept at and why?
5°C to prevent contamination
What are the two types of agar and what do they do?
- Simple media (nutrient agar)- Provides the basic nutrients required by undemanding species of bacteria
- Enriched media- Used to culture bacteria that require additional nutrients
What is blood agar used for and what does it contain?
•Blood agar contains 5-10% blood (normally sheep blood)
•It is used to support the growth of most mammalian pathogens while it can also be used to detect haemolysis
What is chocolate agar used for and what does it contain?
•Contains blood that has been heated to 80°C
•Heating the blood causes rupturing of red blood cells which releases haemoglobin, this adds nutrition from inside the red blood cells to the agar
What are enriched broths, give an example?
•Liquid medium which is selective for a particular bacterium
•No inhibitory agent prevents growth of other organisms
•Enriching conditions allow the growth of the target species, whilst also permitting it to outgrow other species of bacteria which may be present
•Example= selenite broth- specifically used for the growth of salmonella
What is a selective media?
•Allows for the growth of particular bacterium or groups of bacteria
•Inhibits the growth of unwanted species
•Some media will change colour with microbial growth this is an indicator media
What does McConkey agar contain and what does it do?
•Contains crystal violet and bile salts
•Selective for lactose- fermenting enteric bacteria and bile salt- tolerant gram negative bacteria
•Suppresses growth of gram positive bacteria = selective and differential
•Bacteria that produce pink colonies (lactose fermenters)= coliforms. E.g. Escherichia Coli
What does Eosin Methylene Blue (EMB) agar contains and what does it do?
•Selective and differential medium used for isolation and differentiation among members of the Enterobacteriacae- enteric (gut) bacteria
•Inhibits gram positive bacteria thus favouring gram negative bacteria
•Also contains lactose- used to differentiate between lactose fermenters and non fermenters
•Acid produced by the fermentation cause the dye to precipitate on the dye surface producing a green metallic sheen
•Non fermenting enteric bacteria do not produce an acid so their colonies remain colourless or take on the colour of the medium
•Smaller amounts of acid can produce a pink colouration
What is deoxycholate- citrate agar?
•Inhibits the growth of non- enteric bacteria
•Used predominantly in the growth in Salmonella
What does brilliant green agar do?
•Inhibits gram positive bacteria and some gram negative bacilli
•Phenol red serves as a pH indicator
•Colour change to yellow colour as a result of acid production in the fermentation of the lactose and/ or sucrose in the medium.
What is the typical colonial morphology of the Brilliant Green agar?
1.Salmonella: white to red, opaque colonies surrounded by red zones in the medium.
2.Escherichia coli: yellow to greenish- yellow colonies surrounded by intense yellow- green zones in medium
- Pseudomanas: pink to red colonies
- Gram positive bacteria: no growth to trace growth
How to look/ analyse your streak for isolation?
•Examine your plate and make notes…
~Any contamination?
~Too much liquid?
~Colony density too high?
~Try to identify one, big, uncontaminated single colony…
What is the method for the first part of looking at a streak for isolation (forming a broth)?
- In the safety cabinet use a sterile swab to remove your chosen colony
- Add the colony to 4ml of sterile water
- Mix vigorously to ensure the colony mixes to the broth (may be dehydrated)
- Keep your isolated streak plate to one side for use in Gram staining later
What is the method for the second part of looking at a streak for isolation (make a purity plate)?
- Label a nutrient agar plate accordingly (initials, date and purity plate)
- Dip your swab into your broth and perform a continuous streak
- Turn the plate 90°C dip your swab in the broth again and repeat continuous streak
- Turn plate 45°C, repeat continuous streak
- Replace the lid of the plate, secure with bands and incubate for API testing
What is the method for the third part of looking at a streak for isolation (make a sensitivity plate)?
1.Label a sensitivity accordingly
- Dip your swab into your broth and perform a continuous streak
- Turn the plate 90°C, dip your swab in the broth again and repeat continuous streak
- Turn plate 45°C repeat continuous streak
- Whilst the plate is still wet, apply the antibiotic stamp to the plate
- Replace the lid of the plate, secure with bands and incubate to read sensitivity testing.
What is the method and result for antimicrobial sensitivity testing?
- Addition of dics containing antibiotic onto a uniform layer of bacteria
- Incubate 37°C for 18- 24hrs
Result-
•Zone of inhibition if sensitive to AB
•No zone if resistant to AB
What is the method for the gram staining procedure?
- Wash your hands and take a clean microscope slide (clean with industrial methylated spirit and a tissue)
- In the safety cabinet, flame sterilise a clean loop and cool the loop
- Choose an isolated colony and pick it up with the loop and smear onto the slide in a thin layer (can add a drop of sterile water if particularly dry).
- Choose an isolated colony and pick it up with the loop and smear onto the slide in the thin layer (can add a drop of sterile water if particularly dry).
- Air dry the microscope slide and fix the slide by passing through the opposite side through the outer cone of the roaring blue flame (Do not overheat slide!)
- Take the slide to the staining area, wear gloves when staining and cover the slide with crystal violet stain and leave for 1 min
- Pour off any excess, rinse with iodine and allow to act for 1 min and carefully rinse again with distilled water for 5 seconds
- Wash the slide with decolouriser until no more dye comes off then wash with water to avoid too much decolourisation
- Cover the slide with safrinin and leave for 1 min, pour off any excess and then carefully rinse with distilled water
- Blot off any excess water and air dry
- Once completely dry it can be observed under a microscope
What can you observe about the bacteria under a microscope after staining?
Morphology- Have you found Bacillus (Rods) or Cocci (Balls)
Gram staining- gram negative (pink/ red), Gram positive (blue/ purple)
What is bacterial identification?
Recognising an unknown microorganism by determining its membership in one of the groups previously characterised by classification (family, Genus, species)
What can help identify bacteria in bacterial identification?
•Macroscopic features- Shape, size, colour, smell
•Microscopic features- cocci, bacilli
•Staining- Gram staining
•Simple Biochemical tests- Catalase and oxidase tests
Why is bacterial identification important?
•Healthcare
•Epidemiology
•Pharmaceutical industry
•Additional scenarios e.g. Criminal investigations, microbial forensics (investigation of bioterrorism threats), environmental studies
What is Analytical profile index testing (API)?
•Standardised, miniatured version of existing identification techniques
•Used to identify bacteria and yeasts
•Different strips available
•Up to 20 minature Biochemical tests
How do you choose the right strip for API testing?
Orientation tests:
~Gram stain and morphology
~Oxidase
~Catalase
What tests do we use for API testing?
•API 20E- Identification of Enterobacteriacae and other non- fastidious gram negative bacteria
•API Staph- Identification of clinical Staphylococi and micrococci at Genus and species level, gram positive
What is Oxidase- oxidative metabolism?
• Identifies the presence of enzymes used in metabolism and oxidative metabolism
• Test for cytochrome oxidase
• Present in aerobic bacteria
What does the Catalase test do/ test for?
The catalase test tests for the presence of catalase, an enzyme that breaks down the harmful substance hydrogen peroxide into water and oxygen (look for bubbles)
For using the orientation tests of API20E what type will the bacteria be?
Should be gram negative oxidase negative bacilli
What temperature (°C) should the orientation texts be incubated at?
37°C
What are the additional orientation tests?
•NO2, Motility, MCc, OF medium
•Reading after development of tests TDA, VP, IND
How do you set up an API test strip?
•Label the plastic case with your initials and date
•In the safety cabinet, use broth 2 to fill the wells in an aseptic manner
What is the ELISA test?
ELISA is an immunological technique commonly used in medicine and scientific research, it detects the presence of an antibody or an antigen in a sample.
In what ways can the ELISA test be used?
•Tracking disease outbreaks (foot and mouth)
•Test for the presence of antigen in a sample (FeLV)
•Test for the presence of antibodies in a sample (west niles virus)
•Test for hormones in serum (cortisol, T4 thyroid hormone)
What is Feline Leukaemia virus (FELV)?
One of the most important causes of morbidity in cats present worldwide both within domesticated and wild feilds
•Retrovirus
•3 main groups:
~FeLV-A
~FeLV-B
~FeLV-C
