Lab Practical For Bio 245

Front 1

Catalase Test

what is a positive result,
what is being tested for
what
groups of microorganisms are the tests are commonly used to distinguish between.

Back 1

what is a positive result: immediate bubble formation
what is being tested for: catalase (an enzyme that breaks down hydrogen peroxide, in aerobes)
what microorganisms are the tests used to distinguish between: Stapholococci are catalase positive, streptococci are negative

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Oxidase test

what is a positive result,
what is being tested for
what
groups of microorganisms are the tests are commonly used to distinguish between.

Back 2

what is a positive result: area of slide turns dark blue or purple because of dye (chromogenic reducing agent)
what is being tested for: using cytochrome c oxidase as a terminal electron acceptor
what
groups of microorganisms are the tests are commonly used to distinguish between: Enterics (actually all Enterobacteriae) are oxidase negative while other G- bacteria, such as Pseudomonas aeruginosa are positive

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MSA
what property of the medium makes it selective/differential, what groups of microorganisms it selects for or differentiates between

what a positive/negative
result would look like:

Back 3

what property of the medium makes it selective/differential: Saltyness (7.5% NaCl) selects for Staphylococci, phenol red indicator differentiates S. aureus because when it ferments mannitol sugar the acidity changes the color to yellow from red

Front 4

Blood Agar

what property of the medium makes it selective/differential, what groups of microorganisms it selects for or differentiates between

what a positive/negative
result would look like:

Back 4

Presence of blood allows differentiation between different hemolytic activity (which bacteria use to gain iron)
Alpha hemolysis - turn green as it reduces hemoglobin to metheglobin (our teeth were)
Beta hemolysis - Complete lysis (S. pyrogenes) clearing around colonies
Gamma - none (E faecalis), normal growth on red medium

Front 5

Coagulase

what property of the medium makes it selective/differential, what groups of microorganisms it selects for or differentiates between

what a positive/negative
result would look like:

Back 5

what property of the medium makes it selective/differential: plasma is present in tube

what groups of microorganisms it selects for or differentiates between: organisms that synthesize the protein coagulase (such as S. aureus from other Staphylococci)

what a positive/negative
result would look like: positive - plasma jells and doesn't pour

convert prothrombin into its active
form thrombin. In turn, thrombin converts fibrinogen (another blood protein
involved in clotting) into fibrin, which then polymerizes into a mesh of
protein. It has been proposed that fibrin-coated bacteria can escape
phagocytosis, and thus become more virulent.

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MacConkey’s Agar

what makes it selective/differential,

positive/negative result

Back 6

what property of the medium makes it selective: selects for Gram negative (crystal violet and bile salts inhibit G+)
Differential: ability to ferment lactose, neutral red is a pH indicator, acidic products act on bile salts forming precipitate colored with neutral red

and what a positive/negative result would look like: colonies are pink/red if lactose is fermented and a precipitate is formed if it happens quickly

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IMViC
what property of the medium makes it selective/differential,
and what a positive/negative result would look like

Back 7

what makes it selective/differential: for Gram negatives, distinguish between different enterics, results almost unique. Primarily for E. coli and E. aerogenes.
E. coli - (++- -)
E areogenes (- - + +)

positive/negative result: each component has either positive or negative result (usually positive if it turns red, except citrate, which turns blue)
Methyl red and V-P are never the same result

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TSI
what makes it selective/differential,

positive/negative result

Back 8

For gram- enteric pathogens. Identify Salmonella-Shigella organisms
Indicates ability to ferment lactose, sucrose, and glucose and form gas and H2s.
K/A = only glucose fermentation
A/A = lactose and/or sucrose fermentation
K/K = no fermentation, peptones have been catabolized
contains sodium thiosulfate (a substrate of H2S production) and ferrous sulfate for detection of H2S - blackening.
Time sensitive! Must be checked within 18-24 hours or will get false results - too soon and it could be just lactose being fermented causing A/A. Too late and an A/A could become K/A as growth with peptones produces basic products

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Urease
what makes it selective/differential,

positive/negative result

Back 9

The urease test uses a urea agar slant to test for the ability to hydrolyze urea with
the enzyme urease. Urea is the produce of the degradation of certain amino acids. Many
enterics can metabolize urea, but only Proteus (and just a few other enteric genera) are
able to rapidly metabolize urea to ammonia with the urease enzyme. Pink is considered a
positive, orange or yellow negative.

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Liver
stage

Back 10

(exo-erythrocytic cycle),
striated liver cell pattern. purple and pink

Sporozoites infect liver cells (2) and mature into schizonts (3), which rupture and
release merozoites (4).

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Blood stage

Back 11

(erythrocytic cycle),
Looks like blood smear (durh), small purple rings within RBCs, may see purple gameocytes

A), the parasites undergo asexual multiplication in
the erythrocytes (erythrocytic schizogony B). Merozoites infect red blood cells (5).
The ring stage trophozoites mature into schizonts, which rupture releasing
merozoites (6). Some parasites differentiate into sexual erythrocytic stages
(gametocytes) (7). Blood stage parasites are responsible for the clinical
manifestations of the disease.

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mosquito stage

Back 12

(sporogenic
cycle).
Purple circles in mosquito midgut


The gametocytes, male (microgametocytes) and female (macrogametocytes), are
ingested by an Anopheles mosquito during a blood meal (8). The parasites’
multiplication in the mosquito is known as the sporogonic cycle C. While in the
mosquito's stomach,the microgametes penetrate the macrogametes generating
zygotes (9). The zygotes in turn become motile and elongated (ookinetes) (10)
which invade the midgut wall of the mosquito where they develop into oocysts
(11). The oocysts grow, rupture, and release sporozoites (12), which make their
way to the mosquito's salivary glands. Inoculation of the sporozoites (1) into a new
human host perpetuates the malaria life cycle.

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Tapeworm (Taenia pisiformis)
what disease

Back 13

responsible for taeniasis and cysticercosis in humans.
The latter disease, caused by T. solium, is the most common parasitic infestation of the central nervous system
the central nervous system.
Humans become infected by ingesting
raw or undercooked infected meat (4). In the human intestine, the cysticercus
develops over 2 months into an adult tapeworm, which can survive for years. The
adult tapeworms attach to the small intestine by their scolex (5) and reside in the
small intestine (6). Length of adult worms is usually 5 m or less for T. saginata
(however it may reach up to 25 m) and 2 to 7 m for T. solium.

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Filarial nematodes: Wuchereria bancrofti - microfilariae form
what disease

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Lymphatic filariasis is a parasitic disease caused by microscopic, thread-like
worms. The adult worms only live in the human lymph system. People with the disease can suffer from
lymphedema and elephantiasis and in men, swelling of the scrotum, called
hydrocele. Lymphatic filariasis is a leading cause of permanent disability
worldwide.

The microfilariae (produced by adults) migrate into lymph and blood channels moving actively through
lymph and blood (3). A mosquito ingests the microfilariae during a blood meal (4).
After ingestion, the microfilariae lose their sheaths and some of them work their
way through the wall of the proventriculus and cardiac portion of the mosquito's
midgut and reach the thoracic muscles (5). There the microfilariae develop into
first-stage larvae and subsequently into third-stage infective larvae (7). The thirdstage infective larvae migrate through the hemocoel to the mosquito's prosbocis (8)
and can infect another human when the mosquito takes a blood meal (1).

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Schistosomiasis (Schistosoma mansoni) – cercariae and miracidiae forms
what disease

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Schistosoma are the causative agents of schistosomiasis (a.k.a. bilharziosis). If untreated the disease becomes
chronic- can damage internal organs, and impair growth and
cognitive development in children. The urinary form of schistosomiasis is associated with bladder cancer. Globally, schistosomiasis is the second most
socioeconomically devastating disease, after malaria. The disease is most prevalent in Asia, Africa, and South America, specially in areas where suitable fresh water
snails may serve as an invertebrate host. Schistosoma penetrate the unbroken skin. infecting humans are
Schistosoma haematobium, S. japonicum, and S. mansoni.

e (1). Under optimal conditions the eggs
hatch and release miracidia (2; slide available), which swim and penetrate specific
snail intermediate hosts (3). The stages in the snail include 2 generations of
sporocysts (4) and the production of cercariae (5; slide available). Upon release
from the snail, the infective cercariae swim, penetrate the skin of the human host
(6), and shed their forked tail, becoming schistosomulae (7).

Disease signs and symptoms
Mild itching and a papular dermatitis accompanies exposure and invasion by the
parasite. Above all, schistosomiasis is a chronic disease. Many infections are
subclinically symptomatic, with mild anemia and malnutrition being common in
endemic areas. Acute schistosomiasis (Katayama's fever) may occur weeks after
the initial infection, especially by S. mansoni and S. japonicum. Manifestations
include:
• Abdominal pain and diarrhea
• Eosinophilia — extremely high eosinophil granulocyte (white blood cell) count.
• Fever and fatigue
• Hepatosplenomegaly — the enlargement of both the liver and the spleen.
• Genital sores, which increase vulnerability to HIV infection. These lesions may
continue to be a problem after control of the schistosomiasis infection itself.
Occasionally central nervous system lesions occur: cerebral granulomatous disease
may be caused by ectopic S. japonicum eggs in the brain, and granulomatous
lesions around ectopic eggs in the spinal cord from S. mansoni and S. haematobium
infections may result in a transverse myelitis with flaccid paraplegia. Continuing
infection may cause granulomatous reactions and fibrosis in the affected organs,
which may result in
manifestations as bloody diarrhea, or cystitis (inflammation of the bladder) and
ureteritis, which may progress to bladder cancer. Bladder cancer diagnosis and
mortality are generally elevated in affected areas.

Front 16

What type of DNA damage is commonly caused by UV irradiation?

Back 16

UV is bactericidal, mutagenic and carcinogenic on direct contact. These effects are due to the dimerization of pyrimidines, particularly thymine, in the DNA chain. When thymine dimers are formed, the DNA is distorted and the thymines fail to pair with the adenines on the opposite strands, thus causing a mutation.

Front 17

Why don’t the bacteria all die as
soon as they are exposed to UV light for a short amount of time?

Back 17

ultraviolet light has very little power of penetration, wavelength is absorbed and causes changes over time (some mutations are probably fixed - yes repair methods)

Front 18

What is a plasmid? What is transformation? How did we know that our bacteria received the plasmid?
(You do not have to memorize the transformation efficiency equation.)

Back 18

small circular pieces - Plasmid DNA usually contains genes for traits that may confer selective growth advantage under certain conditions. bacteria can transfer plasmids - share beneficial genes.Transformation is uptake of free DNA without cell contact required (as in conjugation). We knew the pBAD plasmid had been received because it encodes the ability to grow on ampicillin and fluoresce when grown on arabinose.

Front 19

How do we determine the temp requirements of bacteria? What type of special medium (if any) do we need to use?
What groups can we categorize bacteria into based on these characteristics?

Back 19

Grow in nutrient agar broth and incubate at different temps and measure turbidity (Optical Density) with spectrophotometer.
Psychrophile - 0-5 C
Mesophile - 20-45C
thermophile > 45 C

Front 20

RapidID test strip: what is it? what are the advantages/disadvantages?

Back 20

It's a set of 20 microtubules with different substrates used for testing bacterial characteristics. Only for medically relevant Enterobacteriaceae (only oxidase-negative and G- bacilli should be tested). Only 4 hours of incubation before reading results. Results vary with interpretation of colors and tests don't always yield the correct results.

Front 21

disinfectants, antiseptics, and antibiotics
Understand the principles behind performing and reading a disc-diffusion assay (such as the KirbyBauer method for determining antibiotic susceptibility)

Back 21

Filter-paper discs containing known concentrations of different
antimicrobial agents are then placed on the plate. After incubation, the presence of a zone of inhibition around the filter-paper disc is noted. The susceptibility of the organism to the antimicrobial agent is determined by reference to a standard chart of typical zone sizes.
The culture medium should be Mueller-Hinton agar, which gives
reproducible zones of inhibition and does not inhibit sulfonamides. Incubation of the Mueller-Hinton plates must be at 37oC for 18-24 hrs

Front 22

Understand the principles behind performing and reading the E-test strips (for determining antifungal susceptibility)
Why is it so hard to develop new antifungal drugs?

Back 22

E test strip has varying concentration of drug so where the zone of inhibition intersects the strip you can read the minimum inhibitory concentration.
Fungi are Eukarkaryotes, so there aren't as many differences in structure to target. Azols target ERG 11, which makes ergosterol. Can prevent it from entering, make more pumps, or change structure.

Front 23

Why did we test our Streptomyces isolates in this lab?

Back 23

We tested it to see if it was capable of producing antibiotics that inhibited the growth of bacteria.

Front 24

What are coliforms? Fecal coliforms? Which do we care about when monitoring water quality?

Back 24

Colliforms are Gram negative rods that ferment lactose. They are enterics so they are oxidase negative and non-spore forming.
Fecal ones are found in the intestinal tract, not environment, so they're what we care about when measuring contamination from potential pathogens.

Front 25

What diseases are Vibrio species associated with?

Back 25

They are associated with foodborne diseases, "infections". Classified into Vibrio cholera infections and non-Vibrio cholera infections. s. Three species, V. cholerae, V. vulnificus and
parahaemolyticus, are well-documented human pathogens. V. mimicus, is a
recognized pathogen with similar characteristics to V. cholerae, except an
ability to ferment sucrose. Other species within the genus are occasional
human pathogens. Vibrio species account for a significant proportion of
human infections from the consumption of raw or undercooked shellfish.

Front 26

Do you expect to find bacteria in food and water? Why or why not?

Back 26

For sure, they like growing on/in things that we like too! Bacteria are all over, and food and water are quite nice mediums for growth. (depends on environmental parameters for growth)
Often they are harmless, but some species are pathogenic.

Front 27

How do we determine the pH, requirements of bacteria? What type of special medium (if any) do we need to use?
What groups can we categorize bacteria into based on these characteristics?

Back 27

Incubate in tubes of nutrient broth and use spectrophotomometer to determine optical density and optimal growth.
Neutrophils (most) between 6.5 and 7.5 pH.
Acidophiles low and alkalophiles high.
Few things grow below 2 or above 10.

Front 28

How do we determine the O2 requirements of bacteria? What type of special medium (if any) do we need to use?
What groups can we categorize bacteria into based on these characteristics?

Back 28

medium with sodium thioglycollate, which is a strong reducing agent
that reacts with O2 and reduces it to H2O. The medium also contains a redox indicator dye called resazurin. Resazurin in the reduced form is colorless. In the presence of oxygen the dye becomes pink.
1st two can be obligate 1.aerobes - bacteria that grow in the presence of oxygen;
2. anaerobes - bacteria that grow in the absence of oxygen;
3. microaerophiles - organisms that require free oxygen, but only in limited amounts (at levels lower than atmospheric O2 concentrations);
4. facultative - organisms that grow either in the presence or absence of free oxygen

Front 29

What are disinfectants?

Back 29

Disinfectants are bactericidal or bacteristatic chemicals which are
generally not safe to use on animal tissue.

Front 30

What are antiseptics?

Back 30

Antiseptics
are chemicals which kill or inhibit the growth of microorganisms and are safe to
use on animal tissue.

Front 31

What are antibiotics?

Back 31

Antibiotics are chemicals produced by one
microorganism which kill or inhibit the growth of another microorganism.