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- routinely done to monitor the prevalence of antibiotic resistant bacteria
- Observe for a trend in order to take precautionary measures
examples of precautionary measures
- development of new drugs
- determining the molecular basis for resistance and modify existing drugs accordingly
Other uses of KB test
- test the antibiotic sensitivity of an organism
- takes away the guessing when choosing antibiotic
- lets you know if it's gram positive or gram neg.
procedures for KB test
- Prepare a pure culture (18-24 hrs) of the sample on a non-selective mediumAdjust turbidity until it is equivalent to the 0.5 McFarland Turbidity StandardWithin 15 minutes of adjusting the turbidity dip a sterile cotton swab into the sample
- streak a lawn of bacteria on Mueller-Hinton agarLeave the lid agar for 3-5 minutes (no more than 15 minutes) to allow plate to dryApply antibiotic impregnated disks on the bacterial lawn
- Important: where the disk drops is where it stays
- Incubate for 16-18 hours at 33 ± 2C unless otherwise instructed
results of KB test
- Antibiotics diffuse out onto the agar
- Concentration of antibiotics decrease as they diffuse further away from the disks
- After incubation, observe for a clearing on the bacterial lawn (zone of inhibition)
how to come to conclusions with results of KB test
- Measure the diameters of the zone of inhibition
- Interpret the results as “resistant” or “susceptible” according to the guideline provided by the NCCLS
- Interpretation of the zone of inhibition is different for each bacteria-antibiotic combination
- zone of inhibition (yellow)
- bacterial growth (orange)
- plasmodium falciparum ring stage
trypanosoma cruzi in blood smear
entamoeba histolytica intestinal lesions
- Pathogen: entamoeba histolytica
- Symptoms: abscesses; significant mortality rate
- Reservoir: humans
- Diagnosis: microscopy; serology
- Treatment: metronidazole
- Causative agent: trichomonas vaginalis
- Found in: semen or urine of male carriers
- SS: Vaginal infection that causes irritation and profuse foul, greenish yellow frothy discharge
- Diagnosis: microscopic ID; DNA probe
- treatment: metronidazole
Where is it identified?
- ID in the blood, CSF
- SS (sleeping sickness), fever, sleepiness, unconsciousness
- Motility due to flagella
- Causative agent: trypanosoma cruzi
- Reservoir: rodents, opossums, armadillos
- Vector: reduviid bug
- Causative agent: giardia lamblia
- SS: most asymptomatic, chronic diarrhea, dehydration, abdominal pain
- ID: by cyst form or trophozoites in stool
- Transmission: contaminated food or water
- Motility: flagella
- Disease: visceral Leishmaniasis
- Causative agent: leishmania donovani
- vector: sandflies
- reservoir: small mammals
- treatment: amphotericin B or miltefosine
- geographic distribution: south asia, sudan, brazil
- Causative agent: balantidium coli
- SS: mostly asymptomatic, diarrhea alternating w/ constipation, blood and mucus in feces
- ID: cyst form or trophozoites
- Motility: cilia
- Causative agent: Plasmodium falciparum
- SS: chills, nausea, vomiting
- vector: anopheles mosquito
- definitive host: anopheles mosquito
- control: bed nets
- treatment: artemisin
Life Cycle of Malaria
- 1. Infected moquito bites human; sporozoites migrate through bloodstream to liver of human
- 2. Sporozoites undergo schizogony in liver cell; merozoites are produced
- 3. Merozoites released nto bloodstream from liver may infect new RBCs
- 4. Merozoite develops into ring stage in RBC
- 5. Ring stage grows and divides, producing merozoites
- 6. Merozoites are released when RBC ruptures; some merozoites infect new RBC and some develop into male and female gametocytes
- 7. Another mosquito bites infected human and ingests gametocytes
- 8. In mosquito's digestive tract, gametocytes unite to form zygote
- 9. Resulting sporozoites migrate to salivary gands of mosquito
- Pathogen: crytposporidium hominis
- SS: self-limiting diarrhea; may be life threatening in immunosuppressed ppl
- Reservoir: cattle; water
- Diagnosis: acid-fast stain; FA; ELISA
- Treatment: oral rehydration
Ascaris lumbricoides eggs
Enterobius vermicularis eggs
Enterobius vermicularis eggs (too)
schistosoma japonicum eggs
schistosoma mansoni miracidiae
Percentage of people infected by helminths
- 1. Ascaris (30%)
- 2. hook worms (21%)
- 3. enterobius (10%)
- 4. schistosome (6%)
- 5. tapeworms (3%)
- 6. flukes-liver, lung (1.5%)
- 7. trichinella (1%)
Name the three groups of parasitic helminths
- 1. trematodes (flukes)
- 2. cestodes (tapeworms)
- 3. nematodes (roundworms)
Scientific name, Disease, Location, Infectivity, Identification, and Treatment of Trematode Parasites
- 1. C. sinensis
- 2. Clonorchiasis (liver disease)
- 3. Japan, Korea, Taiwan, US (Southern Asian Immigrants)
- 4. ingestion of infected undercooked fish
- 5. eggs in feces
- 6. Praziquantel - increases the permeability of the membranes of parasite cells(paralysis); Albendazole - depletes their glycogen stores
Clonorchis sinensis (liver fluke) life cycles
- 1. Embryonated eggs passed in feces
- 2. eggs ingested by snail, go from miracidea to sporocysts to rediae to cercariae
- 3. free swimming cercariae encysts in the skin or freshwater fish
- 4. Metacercariae in flesh or skin of freshwater fish are ingested by human host
- 5. excyst in duodenum
- 6. adults in biliary duct
What is the streak plate method and why is it used?
Method of isolation that allows for the formation of individual colony forming units by reducing the number of bacterial cells with each streaked quadrant.
What is a pure culture and how is it obtained?
- a laboratory culture containing a single species of organism.
- A pure culture is usually derived from a mixed culture (one containing many species) by transferring a small sample into new, sterile growth medium in such a manner as to disperse the individual cells across the medium surface or by thinning the sample manyfold before inoculating the new medium.
What is aseptic technique?
refers to a procedure that is performed under sterile conditions.
What are the cultural characteristics of bacterial colonies?
how do you make a bacterial smear
A Slide that has had bacteria placed on it and that has been treated to cause the bacterial cells to adhere to the slide
Small drop of water scoop small amount of bacteria onto slide and spread in small cirular motionheat fix to adhere the slide
why is heat fixation used
To adhere the bacteria to the slide and to help sterilize the surrounding area of the slide
what is a hanging drop method and why is it used?
method where a drop of a culture is placed directly on a coverslip (there is a circular depressed area in the middle of the slide) allowing the drop of broth to hang in the space of the depression. This technique is useful for thick cells such as protozoans must be examined
Used to examine live organisms
what is Brownian motion
when nonmotile organisms exhibit a dancing or jiggling motion that does not result in traveling any distance this is a specific name for this motion
Why would you perform a simple stain
to be able to examine single cells through a microscope.
Used as general, all purpose stains.
Usually basic dye that will stain the cell membranes are negatively charged.
What are the morphologies of bacteria?
- 1. Cocci
- 2. Bacilli
- 3. Vibrios
- 4. Spirillum/Spirochetes
Steps of Gram Staining
- 1) Choose isolated colony
- 2) Place bacteria on slide in a circular motion
- 3) Heat fix for about 30 seconds
- 4)Place on staining tray
- 5) Flood surface with crystal violet
- 6) rinse with water
- 7) flood surface with iodine
- 8)Rinse with water
- 9)Flood with Alcohol
- 10) rinse with water
- 11) flood Safranin
- 12) rinse- blot slide read with oil immersion lens of microscope- look for gram - and gram +bacteria
steps to acid-fast staining
- Prepare smear. Heat fix.
- Add Kinyoun's carolfuchsin and stain for 5 min. Rinse with water.
- Flood slide with acid-alcohol for 3 min. Rinse with water.
- Add acid-alcohol for 1 min or until no more stain is removed from smear. Rinse with water.
- Counterstain with methylene blue for 1 min.
- Rinse slide with water and air-dry.
Schistosoma life cycle and route of infection
- 1. adult female lay eggs
- 2. eggs reach body of water after being excreted in feces or urine
- 3. eggs hatch into free-swimming larvae -> miracidium
- 4. miracidium penetrates snail
- 5. miracidium reproduces in snail, forming several cercariae
- 6. cercaria are released from snail
- 7. free swimming cercariae penetrate human skin, losing tail
- 8. cercariae travl through circulatory system to intestinal BVs where they mature into adults.
Tapeworms life cycle
- 1. adult female lay eggs in feces or urine
- 2. eggs hatch and release miracidia
- 3. miracidium penetrates snail
- 4. miracidium reproduces in snail -> sporocysts ->cercariae
- 5. cercaria are released from snail
- 6. free swimming cercariae penetrate human skin
- 7. cercariae lose tail during penetration, become schistosomulae
- 8. travel through circulatory system
- 9. migrate to portal blood in liver and mature into adults
- 10. paired audlt worms migrate to mesenteric venules of bowel/rectum (laying eggs that circulate to the liver and shed in stools) and to venous plexus of bladder
Necatur americanus life cycle
- 1. Eggs are passed in the stool, larvae hatch in 1 to 2 days. The released rhabditiform larvae grow in the feces and/or the soil
- 2. after 5 to 10 days they become filariform (third-stage) larvae that are infective .
- 3. On contact with the human host, the larvae penetrate the skin and are carried through the blood vessels to the heart and then to the lungs. They penetrate into the pulmonary alveoli, ascend the bronchial tree to the pharynx, and are swallowed .
- 4. The larvae reach the small intestine, where they reside and mature into adults. Adult worms live in the lumen of the small intestine, where they attach to the intestinal wall with resultant blood loss by the host .
- 5. Most adult worms are eliminated in 1 to 2 years, but the longevity may reach several years.
having both male and female organs in the sameindividual; hermaphroditic.
having the male and female organs in separate and distinct individuals; having separate sexes.
difference between cestodes, nematodes, and trematodes
the anterior, headlike segment of a tapeworm, having suckers,hooks, or the like, for attachment.
one of the segments or joints of a tapeworm, containing completereproductive systems, usually both male and female.
Differential and selective media purpose
- Useful in clinical isolation of enterobacteriaeae
- Media are designed to encourage the rowth of nterics while discourage the growth of harmless bacteria
- Distinguish lactose fermenters such as E. coli from non-lactose fermenters such as Salmonella Shigella
MacConkey agar is designed to select for ___ organisms and differentially stain them for ___ ___
- Gram -
- lactose fermentation
In MacConkey agar, the neutral red dye stains microbes that ___ ___
EMB stands for
Eosin methylene blue
EMB is both a selective and ___ media.
EMB inhibits the growth of ___ bacteria.
EMB provides a ___ indicator distinguishing between organisms that ferment lactose (E. coli) and those that do not (Salmonella)
In EMB agar, organisms that ferment latose display "___ ___," colonies with dark circles.
What rsult will you get with E.coli in an EMB test
Hektoen Enteric Agar is a slective and differential agar primarily used to recover ___ and __ from patient specimens
HEA or HE or HEK
Hektoen Enteric Agar
HE contains indicators of ___ ___ and ___ production.
In HE, salmonella produces ___ colonies, Shigella produces translucent ___ colonies [the agar itself is green], where Black = ___ production, Yellow = ___ production, and Blue-Green = ___ ___
- Lactose fermentation
- non-lactose fermenters
The TSI test is named for its ability to test microorganisms ability to ___ ___ and to produce ___
The TSI slant contains agar, a pH-sensitive dye (phenol red), 1% ___, 1% ___, 0.1% ___, as well as sodium thiosulfate
The slanted slope of this medium provides an array of surfaces that aid either exposed to O2-containing air (aerobic environment) or not exposed to air (anaerobic environment)
bacteria growing on ___ may ferment any or all the sugars, producing a characteristic pattern for each nteric bacteria.
Acids will change the color of phenol red to ___. Position of the color change distinguishes the acid production associtated with ___ fermentation from the acidic byproducts of lactose or ___ fermentation
- C: no fermentation
- 1: neg. for fermentation
- 2. glucose only
- 3. glucose fermentation + H2S production
- 4. Glucose + lactose fermentation + gas
- 5. Glucose and lactose fermentation plus gas and H2S
a self-contained sterile plastic tube with 12 compartments containing distinct media that permit different biochemical tests to be done.
Litmus Milk reactions:
- A. peptonization
- B. Alkaline, lactose Not fermented
- C. lower pH, more acidic
- D. lower pH, more acidic
- E. Lactose fermented, most acidic
paramecium in conjugation