UAMS Microbiology Module 1

Who made the Gram stain?

Christian Gram

What are the reagents for the Gram stain?

Crystal violet (initial stain placed on bacteria, purple or dark blue look)

Iodine (will mix with the crystal violet present in bacteria to help it stick, makes mordant).

Acetone-alcohol decolorizer (usually 1:1, ethanol 95%:acetone)

Safranin (secondary stain, the pink one).

How do you make slides from a colony on an agar/media

Mix with a drop of saline.

Generally, place the drop if saline on the slide and then take a loopful of bacteria from a colony and swirl it around.

What are the methods to fix slides?

Just think heat.

Generally with a slide warmer at 60C

Pass through flame 2-3 times.

Hold at front of incinerator (this part gets really hot so use tweezers).

Flood smear with methanol and

What can happen due to the cells themselves, and not the process of staining?

Some GP organisms, as they age or from antibiotics, will have weaker cell walls and may appear GN as they can’t hold in the stain.

The best results will be on <24hr old cultures from non-inhibitory media (blood agar, since it’s only nutrients).

Overheating may distort some cells as well.

What are the characteristics of agar media?

Agar media: Polysaccharide melts at 100 C; stays liquid until 45-50C -Media may be formed into plates, slants, or deeps.

• Semi-solid: 0.3-0.5 % agar

• Solid: 1-2% agar

What does the streak plate method do?

It’s the 3-4 quadrant streaking on the plates.

Separates & semi-quantitatively enumerates
organisms present in patient specimens or mixed cultures.

Principle: Organisms are separated by spreading them over the
surface of the agar plate.

Each successive section has fewer organisms (section 3 < section 2 < section 1) & organisms farther apart.

Colony-forming-units (CFU): Individual or small groups of cells which multiply to form visible colonies. Isolated colonies appear after incubation.
May be unnecessary to sterilize loop between sections when working with patient specimens.

Interpretation:

• Growth only in the first section = 1+ = Few, sparse, or light

• Growth in the first and second sections = 2+ = Moderate.

• Growth into the third section = 3+ = Many or heavy.

What are the types of media?

Nutrient: grows most kinds of fastidious organisms.

Enriched: Supplemented with various substances (blood, blood by-products, vitamins, yeast extract, carbohydrates, etc.). Grow fastidious organisms (special nutritional requirements).

Enrichment broths: Enhance growth of certain MO while inhibiting other organisms.

Selective: Have antimicrobial substances (e.g., dyes, antibiotics, &
chemicals). Inhibit some microbes while allowing the growth of others.

Non-selective/inhibitory: No antimicrobial substances & allow growth of many organisms.

Differential: Metabolic differences distinguish microorganisms. Identification media are differential.

Nutrient media examples

Nutrient agar

trypticase soy broth

Enriched media examples

Sheep blood agar plate (BAP): Most commonly used medium, Grows many microorganisms

• Sheep BAP: Nutrient base (e.g., trypticase soy
  agar) supplemented with 5% sheep blood.

• Blood agar made with human, rabbit, or horse
  blood is used in some situations
  

Chocolate agar (CHOC): Grows many organisms including fastidious bacteria such as Haemophilus.

• Modified blood agar; made by heating sheep RBC
  to release nutrients or by adding nutritional
  supplements to a nutrient agar base

Enrichment broth examples

Gram-Negative (GN) broth & Selenite broth: Used to isolate Salmonella and Shigella from fecal specimens.

LIM broth & Trans-Vagil broth Used to isolate Group B
Streptococci from vaginal/rectal specimens.

Selective broth examples

Have antimicrobial substances (e.g., dyes, antibiotics, & chemicals). Inhibit some microbes while allowing the growth of others.

• PEA agar is blood agar supplemented with phenylethyl
  alcohol.

• CNA agar is blood agar supplemented with the antibiotics
  colistin and nalidixic acid.

• Gram-positive organisms grow on PEA agar and CNA agar while most gram-negative bacilli do not. (Anaerobic gram-negative bacilli grow on anaerobic formulation of PEA).

Non-selective media examples

No antimicrobial substances & allow growth of many organisms.
Examples: BAP and CHOC

Some medias can be multiple. BAP is both nonselective, enriched, and differential due to growing anything, differing by hemolysis, and containing blood cells.

Differential media examples

Metabolic differences distinguish microorganisms.

• Identification media are differential.

• Examples: Carbohydrate fermentation media & enteric media.

What is the most common temp used in micro?

35C (though the range is 35-37C)

Other temps are used for specific pathogens, there will either be certain incubators for this or one incubator will be used and the temp changed depending on the organisms that need recovering.

What are the other few temps needed for micro?

Yersinia enterolitica is 22-25C

Campylobacter jejuni is 42C

What are the types of atmosphere conditions needed?

Aerobic: Air approximately 21% oxygen. Obligate aerobes require oxygen.

Anaerobic: No oxygen, O2 toxic for strict anaerobes, some anaerobes tolerate low O2 level.

Facultative anaerobes: Grow both aerobically and anaerobically.

Microaerobic (also known as microaerophilic): Organisms require a decreased level of oxygen (5% -10%).

Capnophilic organisms: require an increased level of CO2 (5% to 10%).

• Special CO2 incubators

• Candle extinction jars: Place culture plates & lighted, white candle in jar and seal. Colored or scented candles may be toxic. Before the candle burns out, the flame will use some of the oxygen to produce an atmosphere of 1% to 3% CO2.

Humidophilic microbes: require increased humidity (70% to 80%). Some incubators have built-in humidifying systems or water-filled pans to increase humidity.

*The difference between microaerobic and capnophilic organisms is very small. Just remember the key words decreased and increased. (imagine one coming from the bottom to get increased oxy, and the other going down to get decreased oxy).

What are some other considerations for incubating specimens?

Keep lids lightly screwed on unless stated.

Keep plates upside down (agar in the air) so condensation doesn’t drip on the media and colonies.

What is hemolysins and what are the types produced?

It is damage to the RBCs in the agar.

Gamma: No clearing at all.

Alpha hemolysis: green apple, partial clearing of the agar.

Beta: Full clearing, should be able to see through it.

Another called Alpha Prime is alpha hemolysis surrounded by a zone of beta hemolysis (partial clearing and full clearing around that)

What are ways to disinfect the Micro lab?

Sterilization: Destroys all MO including bacterial spores. Exception: Prions (agents that cause transmissible spongiform encephalopathy). Require special methods to destroy.

Disinfection: Pathogenic organisms eliminated. Viable bacterial spores may be present. Antiseptic: Disinfectant used on living tissue (skin)

3) Physical methods: Usually sterilize

• Autoclaves: Pressure & steam sterilize at 15 pounds per square inch (psi) with Temperature of 121 C. 15 min exposure time sufficient for most items. 30-60 mins for infectious waste

• Dry heat: 160 - 180 C for 1.5 to 3.0 hours. Sterilizes glassware/items not tolerating moist heat

• Filtration: Liquids, air, and other gases can be filtered.

• Radiation (Ionizing & ultraviolet radiation.

Chemical agents: Usually disinfect:

• Alcohols: Ethyl or isopropyl alcohol (60 - 90%)

• Aldehydes: Glutaraldehyde

• Phenolics (chemically modified phenols)

• Quaternary ammonium compounds (“quats”)

• Halogens: Bleach (has chlorine, Sodium hypochlorite (NaClO)). 1:10 dilution recommended for blood spills. Clean area first then disinfect (chlorine
  inactivated by organic matter)

• Iodophores (Disinfectants & antiseptics) Iodine compounds/Povidone-iodine.

What are the different biosafety levels?

Level 1: won’t cause disease in normal healthy adults. Wash hands and wipe down things.

Level 2: routinely used by most labs. Human pathogens not transmitted by inhalation. Coats, limited access, and sharps.

Enterics, Herpes simplex virus, Staph. aureus

Level 3: MO transmitted by inhalation, physically separated area, possible respirators, negative pressure, (our labs level).

Level 4: Extremely contagious and dangerous organisms (Ebola).

Mutualism

Both the bacteria and human benefit.

Like intestinal bacteria, they help digest food and make some Vit K for us.

Commensalism

Human is not affected while bacteria benefits.

Normal flora is often this.

Parasitism

Bacteria benefit at the expense of the human host.

Like Grp A strep.

Opportunistic pathogens

Not normally a pathogen unless the body is weakened (immunoompromised, wounds).

Virulence

Due to a variety of microbial factors (toxins, capsules, enzymes). Virulent microorganisms are more likely to cause disease

Carrier

Will carry the disease and can give it to another.

Usually asymptomatic.

Zoonosis

Animal disease in a human.

What are the human defense mechanisms?

Intact skin and mucous membranes

Mucous membrane cleansing activity: Mucus & cilia

Production of antimicrobial substances

• Lysozyme: Enzyme disrupts cell walls of gram-positives;
  found in tears, saliva, and other body secretions.

• Fatty acids: Produced by sebaceous glands; reduces skin’s pH, which inhibits microbe growth

• Antibodies on mucous membranes. Phagocytosis by PMNs, macrophages, and monocytes

• Host susceptibility factors: Nutritional status, age, stress, genetic makeup, and preexisting conditions (surgery, foreign bodies, etc.).

What happens in the invasion of a body by bacteria?

Attachment to host cells:

• Some bacteria (N. gonorrhoeae use pili (short, filamentous, surface structures) to adhere to host.

• Proliferation: Organisms grow and multiple if they effectively compete with the host and other microbes for nutrients.

Evasion of host defenses:

• IgA proteases produced by some organisms & destroy IgA

• Inhibition of Phagocytosis by Capsule: Protein/polysaccharide coats bacterial cell
  (i) Phagocytes engulf encapsulated MO only
  when specific anticapsule antibodies
  present. (Example: Streptococcus pneumoniae)

• Some microbes ingested but not killed by
  phagocyte.

• Antigenic variation: Some MO (Borrelia) change their
  surface antigens as host responds to MO. Host has to play immunologic “catch-up”.

What kinds of toxins are made by bacteria?

Exotoxins: Extracellular proteins produced by metabolizing bacteria.

• Harm host cells by damaging the membrane or by
  interfering with normal metabolic functions.

• Toxins may be named for their site of action.

• Neurotoxins - nervous system.

• Enterotoxins - gastrointestinal tract.

Endotoxins: Lipopolysaccharide (LPS) part of gram-negative cell wall.

• LPS composed of carbohydrates and lipid A (toxic part).

• Once bacteria die, the toxin is released.

• Can cause fever, shock, bleeding, and death.

How do normal flora help the human body?

Can interfere with pathogens by:

• Producing bacteriocins (antimicrobial substances).

• Reducing a body site’s pH (e.g. lactobacilli in female genital tract).

• Competing for nutrients.

NF of Gastro tract

Anaerobic bacilli (Bacteroides, Clostridium)

Enterococci
Anaerobic cocci

Staphylococcus aureus

Gram-negative enteric bacilli

Yeast (Candida)

Genitourinary NF

Lactobacilli

Diphtheroids

Anaerobic bacilli (Bacteroides, Clostridium)

Streptococci

Anaerobic cocci

Gram-negative enteric bacilli
Staphylococci (S. epidermidis, S. aureus)

Acinetobacter

Enterococci

Yeast (Candida)

Respiratory Tract NF

Staphylococci (S. epidermidis, S. aureus)

Haemophilus

Streptococci (viridans, pneumococci)

Gram-negative bacilli (enterics, nonfermenters)
Enterococci

Anaerobes

Diphtheroids

Spirochetes
Neisseria

Yeast (Candida)

Skin NF

Staphylococci (S. epidermidis, S. aureus)

Diphtheroids

Micrococci

Gram-negative bacilli (enterics, nonfermenters)
Streptococci (nonhemolytic)

Anaerobes

Enterococci

Yeasts and fungi