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Microbiology > Bacteria Classification and Gram stain interpretation > Flashcards

Bacteria Classification and Gram stain interpretation Flashcards

1
Q

Basic Classification of bacteria (3 ways)

A
  1. gram stain (positive or negative)
  2. microscopic morphology
  3. anaerobic or aerobic
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2
Q

Gram stain

A
  1. stain all cells with crystal violet (makes them look purple)
  2. add mordant (iodine), to set/fix the stain
  3. decolourize with alcohol: gram positive still purple, gram negative decolourized/clear
  4. add in safranin: stains clear cells pink (gram positive are pink)
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3
Q

Gram stain

- cell wall structural differences and why stain is different

A
  • bacteria have cell walls with peptidoglyan
  • gram positive bacteria have a very thick peptidoglycan layer - difficult to decolourize with alcohol - stays purple
  • gram negative bacteria have a very thin peptidoglycan layer, plus another cell membrane on the outside of the cell wall - easy to decolourize because it’s so thin
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4
Q

Microscopic morphology

  • different shapes (6); most common
  • ways bacteria divide
A
  • cocci/coccus, coccobacillus, bacillus, vibrio, spirillum, spirochete
  • -> most common are cocci, coccobacillus, and bacillus
  • further classified by the number of planes it divides on
    a) single plane: diplococci (binary fission in pairs, then splits off)
    b) single plane: chains (binary fission along one plane, but doesn’t separate)
    c) two planes: tetrad clusters
    d) multiple planes: grape-like clusters
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5
Q

Creating a smear

  • where does sample for smear come from?
  • how to put on slide (3)
  • fixing the smear (2 ways)
  • gram staining (4 steps)
A

Where from?

  • bacterial culture; OR
  • sputum sample

Put on slide

  • put on a slide
  • add saline
  • let it dry

Fixing the smear:

  • heat the fix (this may destroy some of the human cells); OR
  • methanol fix it (beautiful cells we can visualize)
  • once they’re fixed, it means the bacteria won’t slide off the slide

Gram staining:

  • add crystal violet (purple staining)
  • add iodine mordant to fix the stain
  • decolourize with alcohol
  • add safranin
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6
Q

Visualizing Stains

A

100x oil immersion lens, add 10x ocular lens (looking at 1000x)

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7
Q

Usefulness of gram stain (3 points)

A
  • one of the most useful rapid diagnostic tests in modern microbiology labs
    1. provides basis for empiric antibiotic therapy for bacterial infections
    2. may indicate the need for laboratory procedures not routinely used (could see a fungal growth - and haven’t ordered fungal culture; could see anaerobic culture and haven’t determined it)
    3. helps assess adequacy of a specimen (can see whether lung sputum looks like pus cells or epithelial cells from the mouth - not reflecting bacteria causing pneumonia)
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8
Q

Assessing specimen adequacy

A
  • > 25 per low power field (100x), would reject specimen as it is contaminated with oral pharyngeal flora
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9
Q

Aerobic and anaerobic

- bacterial metabolism

A
  • if organisms can use aerobic respiration, they will do so as they’ll have a net gain of 38 ATP
  • if there is no oxygen, they can switch to anaerobic respiration just to survive, but they won’t gain as much energy (<38 ATP)
  • fermentation: only gain of 2 ATP
  • some organisms can ONLY use one of these forms of respiration (some can only do aerobic, some can only do anaerobic)
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10
Q

Aerobes (4 types)

A

–> tolerate being in oxygen

  • strict aerobes: solely depend on oxygen (aerobic respiration). If no oxygen, they’ll DIE
  • facultative anaerobes: both aerobic and anaerobic metabolism. Vast majority of our normal microbiome and bacteria that cause disease in humans.
  • microaerophilic: depends on aerobic respiration, but if there’s too much oxygen they DIE. Also would DIE if they don’t have any oxygen
  • aerotolerant: uses anaerobic fermentation, but will survive with oxygen
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11
Q

Anaerobes (1 type)

A
  • strict anaerobes: anaerobic metabolism. killed by the presence of oxygen (lacks enzymes that break down oxygen free radicals and hydrogen peroxide)
  • if collecting a specimen from a patient that you suspect is an anaerobe, you must protect it from oxygen exposure
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12
Q

Anaerobic and aerobic in test tubes

A

AEROBES
- strict aerobes: can only grow at top (where there’s lots of oxygen)
- facultative anaerobes: can grow throughout; more efficient at the top with oxygen (38 ATP)
- microaerophilic aerobes: grow at top (where there’s oxygen…but not too far up)
- aerotolerant: can grow throughout, using anaerobic fermentation, but won’t die when they’re exposed to oxygen
ANAEROBES
- strict anaerobes: grow at bottom

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13
Q

Divisions of Bacteria with examples!!!!

A

Bacteria
- Gram-positive
–> Cocci
Aerobic: Staphylococcus spp., Streptococcus spp., Enterococcus spp**

–> Bacilli
Aerobic: Listeria spp, Corynebacterium spp, Bacillus spp. (last two are very common but don’t cause disease very often)
Anaerobic: Clostridium spp**.

  • Gram-negative
    –> Cocci
    Aerobic: Neisseria spp, Haemophilus spp (coccobacilli)

–> Bacilli
Aerobic: E. coli, P. aeruginosa

        Anaerobic: bacteroides spp**
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14
Q

Atypical Bacteria

A
  • Intracellular (have to be within a cell to replicate, but are separate cells - not like viruses): Chlamydia spp, Chlamydophila spp, Rickettsia
  • Lack rigid cell wall (cannot be stained): Mycoplasma spp.
  • Acid fast*/ Partially acid fast**: (acid-fast: extra thick waxy cell wall, gram positive because cannot decolourize with alcohol to further classify we add a second stronger decolourizer. Acid-fast still won’t decolourize. e.g. Mycobacterium spp.) (Partially acid fast: thick waxy cell wall, but not as robust as mycobacterium, acid fast decolourizer will decolourize partially acid fast bacteria. We then use a decolourizer that is in between the acid fast and alcohol, and so they are “partially acid fast positive” e.g. Nocardia spp)
  • Spirochetes (different shape): Leptospira spp, Treponema pallidum.
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15
Q

Staphylococcus spp.

  • classification (gram stain, morphology, division pattern, respiration type)
  • list species and details about each species
A

Gram-positive Cocci in Clusters, aerobic

  1. S. aureus:
    - most virulent staphylococcal bacteria
    - -> strain: MRSA (methicillin resistant staph aureus) - very resistant version
  2. Coagulase negative staphylococci (3 species):
    - far less virulent than s. aureus
  3. S. epidermidis (most common coag neg species)
  4. S. lugdunensis (most virulent coag neg)
  5. S. sapropyticus (causes UTIs)
  • *coagulase test:
  • to differentiate between staph aureus and coagulase negative bacteria (fibrin clots)
  • If it clots, it means it is coagulase positive (and so it’s staph aureus!); if it doesn’t, it’s coagulase negative
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16
Q

Streptococcus spp.

  • classification (gram stain, morphology, division pattern, respiration type)
  • list species and details about each species
A

Gram-Positive Cocci in Chains; aerobic

  • Beta-hemolytic streptococci:
  • -> breaks down RBCs, see clearing around chains
  • -> short-chains
    1. S. pyogenes: Group A Streptococci
    2. S. agalactiae: Group B Streptococci
  • Alpha-hemolytic:
  • -> long chains
  • -> breaks down RBCs partially, see “greening” around chains
    1. Viridans group streptococci (normal oral flora; can cause sub-acute endocarditis). Most common alpha-hemolytic strep.
    2. “gram-positive diplococci”: S. pneumoniae (lancet shaped, cocci pairs, clearing around pair suggesting capsule
17
Q

Enterococcus spp.

  • classification (gram stain, morphology, division pattern, respiration type)
  • list species and details about each species
A
  • Gram-positive Cocci in Chains; aerobic
  • short chains
  • E. faecalis
  • E. faecium
  • Vancomycin resistant E. faecium (VRE) ***most common VRE
  • Vancomycin resistant E. faecalis (VRE)
  • hospitals in Canada used to try to isolate patients with VRE
  • cost isolating is not worth cost saved by preventing infection
  • controversial right now
18
Q

Corynebacterium spp.

  • classification (gram stain, morphology, division pattern, respiration type)
  • list species and details about each species
A

Gram-positive Bacilli in Chains; aerobic

  • **will most likely see these bacteria in non-infectious situations (common and normal on skin)
  • will see if skin isn’t properly disinfected, venipuncture will pick up bacteria on the skin and it will grow in the culture.
  • not very virulent. But it is possible for ppl to get infected (especially immune-compromised ppl)

C. jeikeium: more virulent
C. urealyticum (causes UTIs…rarely)
C. diptheriae (not common because of vaccination)
All other species will be called “diptheroids” - typically normal skin flora

19
Q

Bacillus spp.

  • classification (gram stain, morphology, division pattern, respiration type)
  • list species and details about each species
A

Gram-positive Bacilli; aerobic

B. anthracis: very virulent!!!

  • other species are likely normal skin flora
20
Q

Escherichia coli

  • classification (gram stain, morphology, division pattern, respiration type)
  • list species and details about each species
A

Gram-negative Bacilli; aerobic

  • most common gram-negative bacilli
  • part of larger group of enteric gram-negative bacilli called enterobacteriaceae:
  • -> Klebsiella pneumoniae
  • -> Enterobacter spp.
  • -> Citrobacter spp.
  • -> Proteus spp.
  • -> Salmonella spp.
  • short stubby rods, bipolar staining
21
Q

Pseudomonas aeruginosa

  • classification (gram stain, morphology, division pattern, respiration type)
  • list species and details about each species
A

Gram-negative Bacilli; aerobic

  • not as common as E. coli
  • intrinsically more resistant compared to enterobacteriaceae
  • thinner, longer rods, no bipolar staining
22
Q

Haemophilus spp.

  • classification (gram stain, morphology, division pattern, respiration type)
  • list species and details about each species
A

Gram-negative coccobacilli; aerobic

  • most common is Haemophilus influenzae
  • very small; on slide can see large immune cells, smaller gram-positive diplococci (strep pneumoniae), and teeny tiny coccobacilli
  • have to be very careful techs don’t miss on the slide
23
Q

Neisseria spp.

  • classification (gram stain, morphology, division pattern, respiration type)
  • list species and details about each species
A

Gram-negative diplococci; aerobic

  • strep pneumonia (gram-positive diplococci): cells shaped like 2 lancet cells together;
  • Neisseria (gram-negative diplococci): cells shaped like 2 kidney beans/coffee beans
  1. Neisseria meningitidis (causes meningitis)
  2. Neisseria gonorrhoeae (STI)
  3. Oral pharyngeal cavity has non-pathogenic/less virulent Neisseria spp.
24
Q

Clostridium spp.

A 
Gram-positive bacilli; anaerobic
- thick, box-car-like rods
4 species:
C. tetani: 
C. botulinum
C. perfringens
C. difficile (#1 cause of hospital acquired gastroenteritis/diarrhea). You won't actually get sick with C. difficile unless you've had an alteration of normal microbiota (antibiotic)
25
Q

Bacteroides spp.

A

Gram-negative bacilli; anaerobic
- thin, sickly looking rods (doesn’t like aerobic environment of gram smear)

B. fragilis group is most common Bacteroides group

26
Q

QUIZ Q:
- a gram stain of a skin abscess aspirate shows many pus cells and Gram-positive cocci in clusters.

Which organism is this most consistent with?

a) staphylococcus aureus
b) streptococcus pyogenes
c) escherichia coli
d) clostridium perfringens

A

Staphylococcus aureus

27
Q

QUIZ Q:
- a gram stain of a knee aspirate shows many pus cells and gram-negative diplococci.

Which organism is consistent with this?

a) Escherichia coli
b) Neisseria gonorrhoeae
c) Haemophilus influenzae
d) Bacteroides fragilis

A
  • NOT streptococcus pneumoniae (cause that’s gram-positive)

Neisseria! (gram-negative diplococci: kidney bean shaped!!)

Microbiology (11 decks)

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