microbiolgy lab exam 3

Joseph Lister

any natural and synthetic agent used to inhibit or kill microorganisms

does not kill, temporarily inhibits bacterial growth

kills the bacteria

concentration and length of contact, "make it stronger or leave it longer"

chemical agents used on inanimate objects

used on living tissues

reduce number of microbes present but does not sterilize. Common in food industry. Capable of killing 99.99%, known as a 5-log reduction of a specific bacterial test population, and to do so within 30 seconds

accumulation of bacterial cells that are attached to a surface. Formed in stages, formed in medical settings, and can cause serious infections.

biofilm formation in lungs of cystic fibrosis patients. Bacteria are protected from the immune system and more resilient to medications.

bacteria within plaque produce acid and cause dental cavities.

biofilms in tubs and toilets (red ring)

C1V1=C2V2

chemical produced by a microbe that kills or inhibits the growth of another microbe. (all derivatives originated from products produced by bacterial or fungal species)

based on their molecular structure, spectrum of activity, and mechanism of action

Alexander Fleming

the ability to treat the causative agent of the infection without harming the host.

bacteria have their own enzymes for cell wall formation, protein synthesis, DNA replication, RNA synthesis, and synthesizes of essential meltabilities.

peptidoglycan

over prescription of antibiotics, exposure to sub-optimal levels of antimicrobial agents(not taking full does of Rx), and exposure to microbes carrying resistance genes.

MRSA

methicillin-resistant staphylococcus aureus
- hospital or community acquired
-more deadly than aids

N. gonorrhoae, Streptococcus spp., Enterococcus spp.

spread dilute culture of bacteria over plate. Place sterile filter paper disc containing known amount of antibiotic on plate. Antibiotic diffuses out of disc, creates a concentration gradient.

less concentrated

a zone of inhibition is produced

the more sensitive the organism

sensitive, intermediate, resistant

ZOI, smallest amount of agent needed to inhibit the growth of a microbe. Will vary based on agent and microorganism, determine ZOI and use standardized tables to determine MIC

area of clearing around the antibiotic disk (no bacterial growth), measure the diameter.

inverse

sensitivity

smaller

0 to 20

20 to 45

45 to 80

80 or higher

inhibits, heat

Slower, not suitable for all materials, ash, etc.
-incineration, hot air ovens, used for sterilization of glassware

production of water vapor results in much better transfer than dry. Less time.

enzyme denaturation, protein coagulation

pasteurization, boiling, autoclaving

does not

uses precisely controlled heat to decrease the microbial load found within certain liquids (like milk)

extend shelf life. 63 degrees for 30 min followed by membrane filtration

high temp, short time. 72 degrees for 15 seconds. 5-log reduction, killing 99.99% of the number of microbes in milk. Adequate for destroying almost all mold, yeast, and spoilage microbes.

<20,000 CFU/ml to <10,000 CFU/ml total bacteria

sealed device that uses steam under pressure to kill microbes. By increasing the pressure within a closed vessel, you can raise the boiling temperature of water. Directly proportional to pressure applied.

used in medical waste facilities to sterilize equipment and waste

endospores; prions

archaea species of microbes

thermal death time- time required to kill all microbial cells at a specific temperature. Used in pharma and cosmetic industries.

decimal reduction time, time required to kill 90% of organisms present at a given temperature, common in canning industry.

more

x-rays and gamma rays- shorter wavelength, higher energy.

have enough energy to remove electrons from atoms, creates "free radicals". Can be used for sterilization, but cost of equipment if high and special facilities required for containment. Medical supplies.

UV, visible light, infrared, microwave, and radiowave

lower

electromagnetic radiation that does not have sufficient energy to remove electrons from the outer sell of atoms.

can be used to disinfect exposed surfaces, air/water, can be used to sterilize operating rooms, food preparation areas, etc.

UVA, UVB, UVC depending on wavelength

200nm

UVA

melanin production is up-regulated

complementary base pairing holds two strands together- A and T, C and G. Double helix structure

UV light causes adjacent thymine's to pair- formation of a pyrimidine dimer. "Bulge" in the double helix- must be repaired before replication, if not repaired mutations may result.
-change in the DNA sequence
-repair may not occur if damage is too severe or too long.

photoreactivation. Photolyases are activated upon exposure to visible light to repair the damage. Bind the DNA in the dark and interact with folic acid in the presence of light to repair the damage.

excision repair. Enzymes are not light regulated. Instead of repairing the damage, the stretch of nucleotides around the dimer are removed. Polymerase then adds new bases, and the strand is rejoined by DNA ligase.

healthcare facilities and research labs

lab working areas such as on a surface (within biosafety cabinets), useful for materials that cannot withstand high temperatures. Can be used in water treatment, food safety, disinfection of operating rooms, etc.

rare autosomal recessive disease, extreme sensitivity to UV light, people with XP have defective nucleotide excision repair pathways, children with skin cancer as young as 10.

improper handling and preparation or food-processing and distribution errors

Salmonella spp., Clostridium spp., Camplyobacter jenjuni, Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, Norovirus, Toxoplasma

Salmonella spp.

Clsotridium spp.

Campylobacter jejuni

Staphylococcus aureus

fresh soft cheeses, milk products psychrotolerant

E. coli

Norovirus

Toxoplasma

food

spread plating

30-300

2,097,252

Salmonella and Shigella

selective and differential for enteric pathogenic bacilli

bile salts and dyes (brilliant green) inhibit Gram + and most gram - bacilli

lactose/neutral red and sodium thiosulfate/ferric citrate

reddish/pink colonies, non fermenters are colorless

colonies with black centers

majority of GI pathogens do not ferment lactose, while the majority of intestinal normal flora do ferment lactose

Eosin Methylene Blue agar

selective and differential

gram negative bacteria, Eosin and methylene blue are dyed that inhibit the growth of gram + organisms

lactose fermentation

greenish metallic sheen

pink

Mannitol Salt Agar

selective and differential

Gram + organisms such as Staphylococcus, because they are halophilic

presence of mannitol, a common carbohydrate

yellow color change results from pH indicator in response to acid production

as normal flora of the skin

urea, amino acids, salts, lactic acids, and lipids are secreted through the skin and provide microbes with what they need to grow

low pH, dry and salty conditions make for an inhospitable environment that only certain species of bacteria can withstand

more nutrients

sweat gland and hair follicle

most often begins as a red sore near the nose or mouth which soon breaks, leaking pus or fluid, and forms a yellowish (or honey-colored) scab followed by a red mark which heals without leaving a scar. Sores are not painful but may be itchy and peel.
-Very common in children
-Staphylococcus or Streptococcus infection

ulcers forming under a crusted surface infection, usually on the lower extremities. Deeper skin infections.
-Untreated impetigo can turn into this
-Usually corresponds with poor hygiene
-Higher chances of infection if immunocompromised

the inflammation of one or more hair follicles.
-Common
-Superficial - outer skin layer
-Deep - penetrates the inner dermis layer - usually due to toxin producing strains

spreading infection of skin and tissues immediately beneath the skin
-Redness, pain, tenderness, sometimes accompanied by fever and chills
-Will require antibiotic treatment most of the time

furuncles (boils) and carbuncles

caused by hair follicles, thus resulting in the localized accumulation of pus and dead tissue.
-Individual boils can cluster together and form an interconnected network of boils called carbuncles.
-In severe cases, boils may develop to form abscesses.
-A severe deep tissue infection

•Desquamative rash; caused by toxins released by Staphylococcus aureus
•Causes layers of the skin to detach; followed by peeling away of the detached layers
•Caused by certain toxin - producing strains (not all strains produce toxins!)

•Common but opportunistic skin bacteria
Primarily found in hair follicles and sebaceous glands
•Cause of some types of acne
•Can also cause infection after operations
•Anaerobic
•Gram-positive bacilli

typically group A, Gram + cocci, usually in chain arrangement. Large threat to neonates. Infections can be mild or severe. Toxic Shock Syndrome

diplococci

B

soil and water, gram - bacillus, skin rashes and ear infections, immunocompromised. Green pigment can be seen at infection site.

spore-forming, gram + rods, tetanus, gas gangrene.

gas gangrene- muscle death, shock, amputations

tetanus-cells become vegetative again and release toxins once exposed to preferable environment (commonly associated with puncture wounds). Vaccines

gram + bacteria that arrange in clusters, salt tolerant, healthy people carry, only a problem if it gets into the body

aureas, epidermis, saprophyticus

"golden cluster seed", facultative anaerobe, coagulase positive, grape-like clusters. Has golden-yellow colonies, often with hemolysis when grown on Blood agar.

aureus

ability to degrade hemoglobin causing the lysis of RBCs. Streptococcal and staphylococcal spp. are commonly differentiated by their hemolytic ability.

partial breakdown, leaves a greenish-brown halo around the bacterial growth.

compete hemolysis, area where growth is has a clearing and is almost completely see through

lack of hemolysis, growth appears as usual

used to visualize hemolytic properties. Basic nutrient agar with a small percentage of sheep blood added

tests for the presence of enzyme catalase. add some H2O2 to bacteria on the slide, and bubbles (O2 released) is positive.

all aerobic organisms and most facultative anaerobes. Staphylococcus spp. are catalase positive and streptococcal spp are catalase negative.

causes activation of the human blood clotting system,- converts fibrinogen to fibrin- resulting in the clotting of blood

tests for coagulase enzyme, enzyme is found on the bacterial surface= coats itself in a clot, and hides from immune system.

aureus

selective for gram + bc it has a high concentration of sodium chloride. Differential for mannitol- pH because of pH indicator phenol red, and the

mouth, nose, nasal cavity, sinuses, throat, and larynx. In contact with the air we breathe

trachea, bronchi, bronchioles, and alveoli. Normally sterile due to ciliary escalator

staphylococcal, streptococcal (mostly), Neisseria, and Haemophilus spp.

ciliated cells beat and throw microbes back into the mouth

competition between microbes

viral and some fungal

Bordetella pertussis (whooping cough) and corynebacterium diphtheriae bacterium (diptheria)

Based on the grouping of cell wall carbohydrates; most important are A, B, and D

streptococcus pyrogens- causes strep, skin infections, beta hemolytic, susceptible to bacitracin (antibiotic)

streptococcus agalactiae- can be found in genital tract of women and results in neonatal meningitis/sepsis

enterococcus faecalis, UTI

using agglutination testing

alpha hemolytic, predominant in oral cavity and cause cavities

commonly caused by streptococcus pyrogens, white patches on tonsils, strep throat

Streptococcus pneumoniae

catalase negative, hemolysis, antibiotic resistance/sensitivity

upper

lower

used for the presumptive identification of streptococcus pneumoniae, Os, from other alpha hemolytic streptococci such as streptococcus viridans (Or)

used to differentiate streptococcus pyrogens from other streptococcal spp. S. pyrogens is sensitive Bs to bacitracin while other strep are resistant Br

mitis- salivarius

used for isolation of viridians Streptococci, common cause of dental cavities. Selective- inhibits gram + (except strep) with Crystal Violet. Contains potassium tellurite that inhibits gram -. Differential because of growth differences, that identify species of strep.

Large pale blue-dark blue opaque mucoid colonies with a gum drop appearance.

Blue small colonies with a dome center

Raised convex colonies with a frosted glass appearance

Raised smooth hard colonies that appear "embedded" in the agar

(now enterococcus faecalis) dark blue-black colonies

CO2 rich - cangle jar

digestion, metabolism, immune system

less, less than 10^4 orgnaisms/ml of secretions. B/C gastric acid in stomach and rapid movement of food through the small intestine- peristalsis

significant, colon.

10^11, feces

commensals, mutualistic

K, biotin, folic acid, etc.

residential, pathogenic

the ingestion of contaminated food or water

anaerobes

G- facultative anaerobes (enteric family)

stomach and duodenum (biopsy)

+Stool- presence of mucus, blood, etc. Color and consistency can be absorbed, may require selective and differential media. Salmonella, shigella spp. may require enrichment. Can also look for parasites and test for viral infections
+Rectal swab
+sometimes blood cultures- typhoid fever resulting from Salmonella infection

bile salts and crystal violet inhibit gram positive organisms

pH indicator and lactose fermenters

neutral red. Pink/red= lactose fermenting

bile esculin agar

members of the genus Enterococcus

Bile inhibits the growth of gram-positive bacteria other than Enterococcus and some streptococcus. Many gram - will grow

the ability to hydrolyze esculin. If an organism can hydrolyze esculin in the presence of bile, the product esculin is formed. Esculin reacts with ferric citrate, which turns the slant a dark black or brown color.

hydrolyze esculin, no color change.

light/pale brown

sterile

urethra

shorter urethra

skin contamination upon exit

1000 or 100

inflammation of the urinary bladder

inflammation of the urethra

inflammation of the kidney

Mostly Gram-negative bacilli, especially E.coli. Can be caused by Gram-positives like Staphylococcus spp. or Enterococcus spp.

cystitis more common in females, UTIs are often nosocomial

clean, midstream urine, and culture. Microscopic exam- Presence of cats leukocytes

Fluoroquinolones, Sulfa drugs, Keflex, etc.

Pyelonephritis: Ampicillin, Amoxicillin, Cephalosporins

b/c urine normally becomes contaminated with bacteria during discharge through urethra. wash/wipe then catch.

>10^5 bacteria/ml

´Spinal needle through the skin to the bladder; local anesthesia

•Color
•Odor
•Specific gravity (how diluted)
•pH
•Presence of red blood cells
•Presence of leukocyte esterase
•Proteins, glucose, ketones
•Detection of nitrites
•Think about how many bacteria can reduce nitrate!
•Presence of crystals

UTI

nosocomial infection, via catheterization

E. coli, Proteus, Enterococcus, Pseudomonas, Enterobacter, Serratia, Staphylococcus, etc.

magnesium chloride and potassium sulfate in the media promote pigment production. Pseudomonas spp produce pigments.

isolated colonies surrounded by a blueish greenish zone.

reddish brownish and yellow

Irgasan- antimicrobial agent in the medium that inhibits gram + and other gram - other than pseudomonas.

waste product of protein digestion found in the vertebrae urine.

exoenzyme that hydrolyzes urea to CO2 and ammonia

Phenol red (normally pH 6.8 and orange)

fushia

enterics, Proteus

Proteus