Control of Micro Organisms (test 2) Flashcards
Purpose of microorganism control.
PREVENT INFECTION diseases in humans, animals, & plants
- PRESERVE food & meds
- PREVENT CONTAMINATION of pure culture research
Types of microbial control.
physical
chemical
chemothereapeutic
Words ending in -cide or -cidal.
kills them all (remove the agent: no growth)
Words ending in -stasis or -static.
stops growth (bacteria is still there) -if you remove the agent it will grow back
Sterilization.
destruction of ALL forms of life (includes bacterial spores)
Disinfection.
destruction of PATHOGENS (on objects disinfectant and on ppl antiseptic
Seps.
putrid or rotten
Aseptic.
condition free of contaminating microbes
Septic.
condition with microbial contamination.
Sanitize
reduction of microbial population on and in an object (ex. milk)
Degerm.
removal of microbes from surfaces (sterilized surfaces, typically used industrially)
Factors that determine effectiveness of antimicrobial measures.
- Microbial population
- Environmental factors (organic matter cane protective)
- Times exposed
- Intensity/concentration of control measure
- microbial characteristics (gram +/-)
Which is more resistant gram +ve or gram -ve?
gram -ve
What are the 5 most susceptible infectious agents?
Enveloped viruses Gm+ bacteria Non enveloped viruses Fungi Gm- bacteria
What are the 5 most resistant infectious agents?
Prions Bacterial endospores Mycobacteria Protozoa (cysts) Fungal spores
(the spore formers, etc.)
If you have have death rate of 90% deaths per min. and your starting population is 1 million, how long would you have to apply the antimicrobial to eliminate the pop. of microbes?
after one min. 100 000 after 2 min. 10 000 after 3 min. 1000 after 4 min. 100 after 5 min. 10 after 6 min 1
7 minutes to total destruction
What are the types of physical control methods?
heat filtration radiation drying cold
What is the most effective and most cost effective method of sterilization and disinfection?
heat
Thermal death time.
TIME required to kill a pop’n of microbes at a GIVEN TEMPERATURE
Thermal death point.
TEMPERATURE required to kill a pop’n of microbes in a GIVEN TIME
How does heat kill?
- Destruction of membranes
- Denaturation (and oxidization) of proteins
- Dehydration
How does oxidation effect heats ability to kill?
speeds up reactions; oxygen attaches to different things and destroys different components of the cells
What are the 2 types of heat?
moist and dry
What advantages are there to the use of moist heat?
CONDUCTS HEAT (better penetration of material) Requires LOWER TEMPERATURES
Types of moist heat.
BOILING
PATUREARIZATION
PRESSURIZED STEAM
Boiling.
(kills most in 10-15min; fungal spores and hepA 30 min; bacterial spores 2+ hours)
What is the preferred method of sterilization?
autoclave
What is the pressure that the steam interest the autoclave at?
15psi above atmospheric
What is atmospheric pressure?
15psi
What does psi stand for?
pounds per square inch
What type of things is the autoclave used for?
bedding, utensils, instruments, saline, sol’n
Types of dry heat?
Incineration (direct flame)
Hot air baking
Incineration.
direct flame that is effective in sterilizing
- used on lab utensils (inoculation loop)
- disposal of contaminated objects (gowns, gloves, masks, plastics)
Hot air baking.
- can be used to sterilize
- 160°C for 2 hours kills spores
- used for dry powders, glassware
Filtration.
- physical removal of microbes from liquids or gases (uses a membrane filter)
- used on heat sensitive materials
- degree of removal depends on pore size
What is often needed for filtration?
vacuum to push liquid through the membrane filter
What is filtration often used on?
heat-sensitive materials
like antiserums, and antibiotics
What is the degree of removal during filtration dependent on?
power size
0.45µm, 0.22µm, 0.02µm
What type of filters are used for atmospheric air filtration?
-laminar flow hood
-face mask
HEPA filter
What does HEPA stand for?
high efficiency particulate air
What is the maximum size to be considered it HEPA filter?
Less than 0.5 micrometers
Shorter wave lengths mean…
higher energy
What types of radiation are considered high energy?
Ultraviolet and below is considered high energy
An ion is a…
charged particle
Non-ionizing radiation.
- Primarily UV
- Germicidal for air and object surfaces
- Destroys genetic material
- Poor penetration power
- Special light fixtures in industry, hospitals
- Damaging to tissues and eyes
Ionizing radiation.
- Gamma rays, x-rays
- Good penetration power
- Germicidal by destruction of genetic material
- High energy, very low wavelength
What is ionizing radiation used for?
sterilizing things that can’t be treated with high heat
(foods, drugs (vitamins), vaccines, plastics, sutures
What types of food may be currently irradiated in Canada?
Potatoes Onions Wheat Flour Spices*
What foods are being considered for irradiation?
Meats (1998-Aug2009)
What are the negatives of irradiation?
Generation of O2 free radicals
Destruction of vitamins (A, E, K)
What are some methods of preserving?
- Salting or sweetening
- Drying
- Cold
- Freeze drying
How does salting preserve?
BACTERIOSTATIC
- osmotic pressure shrivels microbes (removes water from around microbe)
- used as a preservative in foods
What is salt used to preserve?
food
How does cold preserve?
BACTERIOSTATIC
- Refrigeration slows growth (5oC)
- Freezing (-5 to -20oC) water crystals may kill some bacteria from inside the bacteria
- Deep freezing (-60 to -80oC)
- Snap Freezing -200oC smaller crystals, high preservation (doesn’t damage the cell membrane as much)
Refrigeration temp.
5 degrees
Freezing temp.
-5 to -20 degrees
Deep Freezing temp.
-60 to -80 degrees
Snap Freezing temp.
-200 degrees
Benefits of snap freezing.
smaller crystals so id doesn’t damage membranes of cells as much
What method has the highest preservation.
snap freezing
Freeze-drying.
LYOPHILIZATION
- freezing causes water crystals, which can be removed under vacuum (sublimation)
- long-term preservation
- astronaut food, lab chemicals, better transport of vaccines.
What type of preservation is more long term?
freeze drying (physical method)
What is freeze drying often used for?
astronaut food, lab chemicals, better vaccine transport
Disinfection.
trying to get rid of pathogens
- antiseptic (on ppl)
- disinfection (on surfaces)
Criteria for antiseptics/disinfectants.
- Must be able to KILL (or SLOW GROWTH of) microbes
- Should be NON-TOXIC to humans & animals
- Should be SOLUBLE IN H2O & have a long SHELF LIFE
- Should PENETRATE well & not corrode instruments
- Should NOT COMBINE with organic matter such as BLOOD or faces
- OBTAINABLE & INEXPENSIVE
- other considerations (25-37 temp, pH=7), bacteria type, type of surface
Who oversees all health-related issues in Canada?
Health Canada
Regulatory body of antiseptics and disinfectants.
Therapeutic Products Directorate (TPD)
What are the guidelines used by the TPD?
Food and Drug Act and Regulations
What tests are used to know what chemical works best against different microbes?
DISK DIFFUSION TEST (not quantitative, used in teaching labs)
USE-DILUTION TEST (quantitative, used by companies, hospitals)
Which test is quantitative for testing chemical ability to fight microbes?
Use-dilution test
Disk diffusion test.
Method:
- disks of filter paper soaked in chemical disinfectant / antiseptic - soaked disks are placed on agar plates previously inoculated with bacteria of choice - size of the ‘clear zone’ around the disk indicates effectiveness of chemical
Down falls of disk diffusion test.
- not quantitative
- doesn’t tell you if it is killing the bacteria
Use-Dilution test method.
METHOD: metal spheres are SOAKED IN BACTERIA being tested, spheres are placed in wells CONTAINING CHEMICAL being tested, how much bacteria grow in the wells indicates effectiveness, test can be AUTOMATED for testing many chemical agents against many bacteria
Turbidity.
measure of relative clarity
Is the use-dilution test quantitative, or bactericidal?
- QUANTITATIVE
- test for BACTERICIDAL by diluting and seeing if anything grows
Draw backs of disk diffusion.
1) Controlled conditions
- no organic matter
- no temp or pH variation
2)Does not test toxicity of chemical
How is the effectiveness of chemicals compared?
Phenol coefficient (PC)
(old method, has draw backs)
- compares substance in terms of how effective it is to the chemical phenol
What is a phenol coefficient?
see how effective phenol is to other chemicals in killing microbes (eg. 133.0 means chloramine is 133X more effective than phenol against staph aureus, because phenol is 1)
If your phenol coefficient for a given chemical was less than phenol (1) then you would have to…
calculate how much more chemical would be required kill a given bacteria.
PC
phenol agent
Germicidal action of chemicals.
1) Disruption of plasma membrane
2) Denaturation or inhibition of protein
3) Inhibition of membrane or protein synthesis
How does phenol work?
denaturing protein at hight concentration, (low concentration and chemical derivative disrupt cell membranes)
What is phenol effective against?
Gram +ve bacteria
- caustic to skin so less toxic derivatives developed
Phenol chemical make up.
6 carbon rings (benzine) attached to OH
Bisphenol is a _______ of plastic manufacturing, _____ against bacteria (especially gram positive).
byproduct
very effective
Another name for Bisphenol.
Triclosan
Types of phenol derivatives.
Chlorehxidine
Triclosan
Chlorehxidine.
in surgical scrubs and mouth wash
Triclosan.
in antibacterial soaps and toothpaste (safety currently under review)
Halogens.
Highly reactive elements (free electrons)
- primarily CHLORINE and IODINE
How does chlorine work?
oxidizing proteins, poking holes in membranes
What is Chlorine effective against?
most microbes (bacteria, viruses, fungi, protozoa)
What is another name for bleach?
sodium hypochlorinate (NaOCL)
What is bleach used for?
water treatment (1/2 teaspoon in 8L of water), equipment disinfection, food processing
How does iodine work?
killing microbes by inactivating
What is iodine effective against?
most microbes (bacteria, viruses, fungi, protozoa), better than chlorine
What are the drawbacks?
- stains
- smells
- not stable
What is used to make iodine more stable and less smelly?
tinctures (combined with alcohol; iodophors (iodine linked to detergents)
Iodophors.
iodine linked with detergents for more stability and less smell
What is iodine used for?
prep prep, food processing (5 drops in 1L of water)
Halogens are easily ____, so they wear out.
inactivated
Why might you need higher concentrations of halogens then indicated?
they easily inactivate (loosing potency)
Once infected how to we kill the microbes?
- IMMUNE SYSTEM
- CHEMOTHERAPEUTICS (because microbes have different structures and metabolisms we can target them)
Chemotherapeutic agents
any chemical (antimicrobial) used to treat infections in the body - usually synthetic (most selective, kill organism without harming host)
Selective Toxicity.
selective to the organism without effecting the hose
Antibiotic.
Antimicrobial agent produced by microorganisms (usually NATURAL ORIGIN, inhibits other microbes in SM AMOUNTS)
Natural origin of aspirin.
willow bark
Natural origin of quinine.
quint tree bark
Natural origin of streptokinase.
Streptococcus bacteria
Natural origin of Hirudin.
leeches
Natural origin of penicillin.
penicillium fungus
Many antibiotics are from
bacteria/ fungi
Spectrum of activity.
the range of microbes the antibiotic is effective against
eg.
Erythromycin: Gram +ve (narrow)
Tetracycline: broad spectrum
What is an example of a broad spectrum antibiotic?
Tetracycline
What is an example of a narrow spectrum antibiotic?
Erythromycin (gram +ve)
Why not use broad spectrum all the time?
If you know exactly what bacteria its best to use narrow spectrum
Broad spectrum contributes to resistance, and kills normal flora
What are the modes of action antimicrobial activity?
- Inhibitions cell wall (membrane) synthesis
- Inhibition of protein synthesis
- Injury to plasma membrane
- Inhibition of nucleic acid synthesis
- Inhibition of essential metabolite synthesis
What are some examples of chemicals as antimicrobial agents?
Sulfonamides
Isoniazid
Quinine
Sulfonamides.
- ex. sulfanilamide (SFA)
- 1st significant chemotherapeutic agent
- isolated form red closing die in 1935
- broad spectrum (gram -ve and +ve)
How do sulfonamides work?
- competitively inhibit folic acid synthesis, as they mimic folic acid precursor PABA (which is required to make folic acid)
- ## works because bacteria require folic acid for synthesis of nucleic acids
Use of Sulfamethoxazole.
UTI’s
Use of Sulfisoxazole.
vaginal infections
Isoniazid (aka isotonic acid hydrazide).
- activated to toxic forms of catalase in bacteria
- inhibits mycolic acid production
- used against TB and Leprosy
What is used to treat TB and leprosy.
Isoniazid
How has TB become resistant to isoniazid (INH)?
- catalase breaks down H2O2, and activates INH to toxic form
- TB down regulates catalase (which would have been activated to make toxic form of INH) and up regulates hydroperoxdase (to neutralize access H2O2 that resulted from less catalase)
Quinine.
- antimalaria
- and to protozoa DNA (plasmodium) and inhibits reproduction
Microbial wars.
war among bacteria; they make compounds to outcompete bacteria or fungi
What are the 4 major sources in order of antibiotics.
1) Streptomyces bacteria
2) Synthetic
3) Gram +ve bacilli
4) Penicillium Fungus
What are some examples of ‘parent’ molecules of antibiotics?
- Penicillin
- Cephalosporin (related to penicillin)
- Streptomycin
- Chloramphenicol
- Tetracycline
Hx of penicillin.
discovered by accident by Alexander Fleming in 1928 (wound up in his petri dish)
- wasn’t isolated till the 30’s
In Penicillin class where is the antibiotic derived from?
Penicillium mold
What is the critical structure of Penicillin?
beta-lactam ring (nucleus)- inhibits peptidoglycan synthesis (blocks crosslinking of carbohydrates to protein)
What does the ‘nucleus’ of penicillin refer to?
the business end of critical structure of thepenicillin
What are the advantages to the penicillin derivatives?
- INCREASED BIOAVAILABILITY
stomach acid resistant, less sticking to food, increased absorption greater 1/2 lives
What are the disadvantages of penicillin derivatives?
LESS EFFECTIVE (decreased spectrum) ORGAN DAMAGE (stomach, kidney, liver)
What are the shortcomings of Penicillin?
1) Narrow spectrum
2) Anaphylactic reactions
3) Antibiotic resistance (penicillinase (beta-lactase) degrades beta lactam ring)
What does penicillinase do?
alters the chemical structure of sodium penicillin G by breaking the C-N bond and binds to water forming sodium penicilloic acid
Cephalosporin.
isolated from a sea water fungus
- similar to penicillin (modified beta-lactam rings)
- inhibits cell wall synthesis
- substitute for penicillin (allergic reactions or resistance)
- 3rd generation developed to work better against gram -ve (named Ceph- or Cef-
Streptomycin.
- isolated from streptomyces bacteria
- member of the aminoglycosides family
(amino groups on carbohydrates) - bind and inhibits ribosomes
- toxic / not well absorbed
- effective against intestinal infection and as a topical ointment (in Neosporin)
What do the less toxic derivatives of Streptomycins all end in?
-mycin
Chloramphenicol.
- isolated from Streptomyces bacteria
- inhibits protein synthesis
- broad spectrum
- very tissue invasive
- effective against meningitis (nerves)
What are the significant side effects of Chloraphphenicol?
- aplastic anemia (relatively rare)
- gray syndrome in newborns (due to inability to metabolize; leads to low BP and death)
Aplastic anemia.
RBCs are being destroyed
Tetracycline.
- isolated from Streptomyces bacteria
- inhibits synthesis
- broad spectrum
- drug of choice for STDs
Side effects of tetracycline.
- stunted bone growth in children
- discolouration of teeth
What do all derivatives of tetracyclines end in?
-cycline
How do you test the effectiveness of antibiotics?
1) Diffusion Method
- Kirby-Baurer test
- E-test
2) Broth Dilution Test
Describe the Kirby-Bauer test (specific to antibiotics).
- Filter paper soaked in antibiotic
- Paper placed on plate with growing bacteria
- Zone of inhibition indicates effectiveness
Is the Kirby-Bauer test expensive? quantitative? show whether it is bactericidal?
expensive? – no
quantitative? Semi-quantitative. Measured across the zone. Not that accurate so not considered a quantitative technique.
show whether it is bactericidal? No – no way of knowing just by looking if you have dead bacteria or if it has just been inhibited
Describe an E-test.
- Plastic coated with a gradient of antibiotic
- Strip placed on plate with growing bacteria
- Where bacteria touch strip tells you the minimum inhibitory concentration (MIC) of antibiotic
- Plate of red coloured agar – could be pH or blood
Pear shaped zone of clearing - Gradient “ zone of clearing edge touches strip. Look at the number, it is somewhere around 0.12.
- Gives you a general estimate of the minimum inhibitory concentration to be bacteriostatic.
Is the E-test quantitative? does it show bactericidal?
more quantitative than Kirby-Bauer test
- no way to tell if it is bactericidal
Broth Dilution test.
Antibiotic is diluted into wells (tubes) containing broth inoculated with bacteria
Let bacteria grow
Lowest conc of antibiotic showing no growth =
What does MIC stand for?
minimum inhibitory concentration
- lowest concentration of antibiotic showing grown in broth dilution test
Same technique as _______ test.
eusdilution test
Is the Broth Dilution test expensive? quantitative? bactericidal?
expensive? yes
quantitative? yes, very accurate
bactericidal? yes, but not on the first go
