Lab Practical 2

u know

simple stain

done to see morphologies and arrangements

Capsule staining

a differential stain that uses acidic/basic dyes to stain background and bacterial cells

Gram stain

differential stain, gram-positive (purple) and gram-negative (pink)

Acid-Fast stain

differential stain,used for mycobacterium, red = positive, blue = non-acid-fast bacteria) 

Endospore stain

special stain used for endospores, (Bacillus, Clostridium)

coccus

round/spherical shape

bacillus

rod shape

vibrio

slightly curved

Spirillum

spiral without movement

Spirochete

spiral with movement

Pleomorphic

“many shapes” bacteria that vary in shape 

Diplococcus

groups of 2

Streptococcus/bacilli

chains of cells

Tetrad

groupings of 4

Sarcinae

Clusters of 8

Staphylococci

clusters of cells

Borrelia burgdorferi

gram negative, Spirochete, causes *Lyme disease*

Treponema pallidum

gram negative, Spirochete, causes Syphilis

Leptospira interrogans

gram negative, Spirochete, causes Leptospirosis

Gram-positive

2 layers: Thick peptidoglycan and plasma membrane, Teichoic Acids, Exotoxin, include: Staphylococcus, Streptococcus, Bacillus, Clostridium, Corynebacterium, and Listeria

Gram-negative

3 layers: Outer membrane, thin peptidoglycan, plasma membrane, Lipopolysaccharide LPS, Endotoxin, includes Escherichia, Haemophilus, Neisseria, Salmonella, and everything else

Acid-Fast

Thin peptidoglycan, Mycolic Acid - Waxy, includes Mycobacterium

Mycoplasma

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No cell wall, only plasma membrane, includes *Mycoplasma pneumoniae*

Bacillus anthracis

gram positive, streptobacillus, causes antrax

Bacillus cereus

gram positive, streptobacillus, causes food posioning

Clostridium botulinum

gram positive, bacillus, causes botulism

Clostridium difficile

gram positive, bacillus, causes C-diff (normal flora, nosocomial)

Clostridium perfringens

gram positive, bacillus, causes gas gangrene and food poisoning

Clostridium tetani

gram positive, bacillus, causes tetanus

Listeria monocytogenes

gram positive, bacillus, causes listeriosis

Corynebacterium diphtheriae

gram positive, bacillus, v-shape arrangement, causes diptheria

Lactobacillus acidophilus

gram positive, bacillus, normal *aginal flora; metabolizes sugars into lactic acid+ maintained acidic pH*

*Bordetella pertussis*

gram negative, coccobacillus, causes pertussis (whooping cough)

Escherichia coli

gram negative, bacillus, causes food poisoning

Escherichia coli O157:H7

deadly strain of E. coli

Haemophilus influenzae

gram negative, bacillus and pleomorphic, causes pneumonia, epiglottitis, meningitis, respiratory infractions, and pinkeye

Klebsiella pneumoniae

gram negative, bacillus, opportunistic, nosocomial

Pseudomonas aeruginosa

gram negative, bacillus, opportunistic, nosocomial

Salmonella typhi

gram negative, bacillus, causes typhoid fever

Enterobacter aerogenes

gram negative, bacillus, Opportunistic, nosocomial infections, normal flora

Helicobacter pylori

gram negative, spiral, causes peptic ulcers

Mycobacterium leprae

acid fast, bacillus, causes leprosy (Hansens disease)

Mycobacterium tuberculosis

acid fast, bacillus, causes tuberculosis

Neisseria gonorrhoeae

gram negative, diplococcus, causes gonorrhea

Neisseria meningitidis

gram negative, diplococcus, causes meningitis

Staphylococcus aureus

gram positive, staphylococcus, causes MRSA “Methicillin Resistant Staphylococcus aureus”, opportunistic,nosocomial

Streptococcus pneumoniae

gram positive, diplococcus, causes pneumonia and meningitis

streptococcus pyogenes

gram positive, streptococcus, causes strep throat

Vibrio cholerae

gram negative, virbio, causes cholera

Organisms used in endospore staining lab?

*Bacillus cereus* and *Escherichia coli* 

Out of the two organisms in the endospore lab which forms endospores?

Bacillus cereus

Staining properties for endospore lab?

Primary stain - Malachite green

Decolorization - water

Counterstain - Safranin 

Two genus that form endospores

Bacillus and Clostridium (both bacillus)

All endospores are gram-

positive

Which culture would have endospores: fresh culture or old culture?

Old culture (bacteria don’t want to form endospore only do it when there is no resources available)

Organisms used in Acid-fast staining lab?

*Mycobacterium nonchromogenicum* and *Staphylococcus epidermidis*

Out of the two organisms in the acid-fast lab which would stain red (is acid fast)?

Mycobacterium nonchromogenicum

What color would *Staphylococcus epidermidis* stain (non-Acid-fast)?

blue

Staining properties for Acid-fast staining?

Primary stain - Carbol fuchsin

Decolorization - Acid Alcohol

Counterstain - Methylene blue 

What do Acid-fast bacteria contain in their cell wall?

Mycolic acid - very thick and waxy

All Acid-fast bacteria have

bacillus shapes

Special Acid-fast medium

Lowenstein-Jensen agar, has malachite green 

Two diseases caused by Acid-fast bacteria:

leprosy (Hansen's disease) and tuberculosis 

(Acid-Fast) Hot/ZN Method what we used, created by Franz Ziehl and Friedrich Neelsen

what we used, created by Franz Ziehl and Friedrich Neelsen

What was the source of heat for the acid-fast?

Steam bath

Other Acid-Fast method

Cold/Kinyoun method

What does ELISA stand for?

Enzyme-Linked ImmunoabSorbant Assay 

(ELISA) What disease did we test for?

HIV

Our three lines of defense

First: skin

Second: nonspecific

Third: specific T-cells (very important for our immune system, stimulate B-cells to produce antibodies), B-cells (antibodies)

Two types of ELISA

direct and indirect

Indirect ELISA

what we used, detects antibodies

Direct ELISA

detects antigens

Equipment for ELISA

microtiter plate (96 wells), pipet, simulated antigen, positive/negative controls, simulated antibody, simulated enzyme, simulated chromogen

How many patients did we test (ELISA)

6 patients (A-F)

(ELISA) 2 differences from the demo and real life:

IRL microtiter plates are coated, we have to add the simulated antigen

IRL they also incubate and wash the plates, we skip those steps (5 hours vs 15 min)

Define pathogens

small disease-causing agents

Examples of pathogens

viruses, bacteria, protozoa

ELISA results

dark color: positive

No color: negative

Light color: weak positive

Bioremediation

the use of microbes to clean up pollutants  

Bacteria used in bioremediation lab?

Pseudomonas culture broth

Fungi used in bioremediation lab?

Penicillium culture broth

(bioremediation) What kind of oil did we use?

motor oil

What else is added with the microbes?

Fertilizer (nitrogen and phosphorus) 

Our three stations:

Group A - Visual (test tubes)

Group B - Open Ocean (Jars)

Group C - Shore (Petri dish)

probiotics

a microorganism introduced into the body for its beneficial qualities

What organism were we looking for?

Lactobacilli

Three yogurts we used

Activia, ratio protein, and stani 

Procedure (yogurt lab)

1. Clean work area well with bleach and dry with paper towels
2. Look at pre-streaked Lactobacilli MRS agar plates and record observations


1. Create a smear and heat fix sample from a Lactobacilli MRS agar
2. The instructor will divide the class into Gram staining and simple staining groups
3. Allow the slides to air dry
4. View under microscope
5. Determine if lactobacillus or other bacteria are present in the culture

How many wells does the microtiter plate have?

96 wells

and the band played on disease

AIDS (acquired immunodeficiency syndrome)

and the band played on etiologic agent

HIV (human immunodeficiency virus)

and the band played on family

retroviridae

and the band played on genus

lentivirus

and the band played on species

HIV 1, HIV 2

beguinning of US AIDS epidemic

1981

san fransisco gay district

castro district

and the band played on - what media labeled virus

gay cancer, gay pneumonia, GRID

Index case

Gaetan Dugas

4-H

hemophiliacs, homosexuals, heroin, haitains

what is factor VIII

blood clotting protein