Microbiology Labs 1

1
Microbiology Labs 1 2

• Introduction to Microbiology

2
What organism is this, and what are some features
of the organism?

• Basic Biology of Genus Staphylococcus
• Gram positive cocci in clusters
• Nonmotile
• Catalase Positive (used to distinguish it from
  Streptococcus)
• Major Pathogen Staphylococcus aureus
• Staphlyococcus Aureus
• Characteristics to distinguish S. Aureus from
  other staphylococci
• Coagulase positive
• Thermostable nuclease positive
• Ribitol in the teichoic acid
• Virulence Factors
• No one factor is decisive for virulence
• Protein A, an IgG binding protein
• Fibronectin, fibrinogen and collagen binding
  proteins
• Hemolysins
• Enterotoxin, heat stable at 100 for 30min!
• Exfoliative toxin (Scalded Skin Syndrome)
• TSST-1, a super antigen (Toxic Shock Syndrome)
• Regulation

3
What organism is this, and what are some features
of the organism?

• Basic points of Streptococcus
• Biology
• Gram positive cocci in chains
• Nonmotile
• Facultative organism, but it can only ferment.
• Streptococci cannot make cytochromes, so they
  lack an electron transport chain.
• Catalase negative (used to distinguish from Staph
  species)
• Classified based on hemolysis on blood agar
  plates (BAP)
• Note Staphylococci are very round, while
  streptococci are elongated with the long axis
  perpendicular to the planes of division. Strep
  pneumonia (the pneumococcus) often has shorter
  chains than other streptococci, with pairs of
  organisms predominating. It is lancet-shaped and
  often has a prominent capsule, observable as a
  clear, refractile halo around the bacteria.

B-hemolysis on blood agar
4
What organisms are these?

• Strep pneumoniae Strep Pyogens

5
What organism is this, and what are some features
of the organism?

• Neisseria
• Basic points (since Neisseria is covered in depth
  elsewhere)
• Biology
• Gram negative cocci in pairs.
• Looks like two kidney beanskissing.
• Neisseria is among the few Gram negative cocci.

6
What organism is this, and what are some features
of the organism?

• Bacillus
• An example of a large Gram positive rod
• Often stains irregularly
• Often indistinguishable from Clostridium by gram
  stain

7
What organism is this, and what are some features
of the organism?

• Escherichia Coli
• An example of a typical gram negative rod.
• Bipolar staining may be present. Generally, it
  is difficult to distinguish between species of
  Gram-negative rods by Gram stain.

green sheen on EMB
8
All cocci are Gram-positive except?

• Genus Neisseria aerobes (gram-negative
  diplococci, kissing kidney beans)
• Genus Veillonella anaerobes (gram-negative
  cocci)
• Any normal Gram-positive cocci which are aging or
  have been grown in an acid environment can stain
  Gram-negative.

9
What is an important general principle to
remember about rods and their Gram-stain?

• Some rods are Gram-negative, and some are
  Gram-positive.
• The Gram-staining reactions of the important
  genera must be memorized.

10
What are spores? How do you stain for them? What
are two-medically important bacterial species
that have spores?
B
A

• A is Clostridium tetani with its spores (head of
  the club/q-tip)
• B is Bacillus Anthracis (red) with its spores
  (green).
• Some bacteria produce spores, specialized
  structures that enable the organism to survive in
  a variety of hostile environments.
• Because spores are generally impervious to most
  dyes, they are usually unstained when the
  bacteria are subjected to Gram-stain.
• However, spores can be stained using malachite
  green. This stains the spore, and the organism
  is then counter-stained with crystal violet.
• The two medically important bacterial species
  that produce spores are Clostridium and Bacillus.
  Note the appearance and location of spores in
  the Gram stained and spore stained smears of
  Clostridium tetani.

11
What are acid-fast bacteria? There are only two
genera that are acid-fast, which ones are they?
Which special stain do you use to identify
acid-fast organisms?

• Some bacteria have cell walls that contain high
  concentrations of waxes and lipids. This makes
  the bacteria resistant to staining and, once they
  are stained, to decolorization, even by solvents
  containing organic or inorganic acids.
• Such organisms are characterized as acid-fast and
  are identified by special staining techniques.
• Only two genera of bacteria are acid-fast the
  genus Mycobacterium and some members of the genus
  Nocardia.
• You will learn to do the Kinyoun modification of
  the Ziehl-Neelsen stain, which is used to
  identify acid-fast organisms (primarily
  mycobacteria) during the laboratory exercise
  entitled Agents of granuloma.

12

• CASE 1 A 70 year old man developed the sudden
  onset of fever, chills and right flank pain. He
  had been in general good health but had noted
  increased urinary frequency for several months
  and mild dysuria for several days. Physical
  examination revealed right flank tenderness and
  prostatic hypertrophy. The diagnosis was made
  from the Gram stain of a clean catch of
  mid-stream urine and confirmed by culture.
• Examination of the Gram stain revealed
  Gram-negative rods.
• What is the most likely bacterium?

• Answer E.coli. Uropathogenic e.coli.
• (Classic UTI)

13

• Case 2 A 55-year old woman with a long history
  of rheumatoid arthritis noted a day or two of
  increased discomfort in her left knee followed by
  the abrupt onset of fever, swelling, and warmth
  of the entire knee. She interpreted this as an
  exacerbation of her rheumatoid arthritis and
  sought medical attention. Aspiration of the knee
  yielded cloudy fluid with pronounced
  luekocytosis, low sugar, and high protein.
  Examination of the Gram stain established the
  diagnosis of septic arthritis and permitted the
  institution of the appropriate antibiotic therapy
  for the specific causitive bacterium.
• Gram stain revealed Gram-positive cocci in
  clusters.
• What is the most likely bacterium?

• Answer Staphylococcus Aureus
• Note Staphylococcus aureus will be the pathogen,
  but you may have the presence of non-pathogenic
  staphylococcus epidermidis due to its ubiquity
  and tendency to contaminate samples.

14

• Case 3 A 24-year old man was well until three
  months after splenectomy for traumatic rupture of
  the spleen. Three days before admission, he
  noted the onset of fever, chills, pleuritic chest
  pain, and cough productive of purulent,
  rust-colored sputum. On admission, his physical
  findings and chest x-ray established the
  diagnosis of consolidated right lower lobe
  pneumonia. Gram stain and culture of his sputum
  were non-diagnostic, but his blood culture was
  positive within 24 hours. The Gram stain was
  made from the liquid medium of his blood culture.
  Examination of this Gram stained slide, together
  with the clinical history, permitted his
  physician to make a presumptive diagnosis and
  initiate appropriate antibiotic therapy.
• The Gram stain demonstrated Gram-positive cocci
  in chains.
• What is the most likely bacterium?

• Answer Streptococcus pneumoniae (a.k.a.
  Pneumococcus)

15

• Case 4 A 43-year old man presented to an
  internal medicine clinic for evaluation of
  anemia. He had had poor appetite and weight
  loss, fatigue, and fever for 3 months. He was
  prescribed iron at an urgent care center one
  month ago, but had not improved. Further history
  indicated rheumatic fever as a child, a known
  cardiac murmur, and no recent dental procedure.
  On exam he was in no distress but had a
  temperature of 101.6, pulse of 96, and blood
  pressure of 152/62. He had subconjunctival
  petichiae, and the cardiovascular exam revealed a
  bounding pulse, a carotid thrill, a III/VI
  systolic ejection murmur radiating to the
  carotids, and a II/VI diastolic murmur along the
  left sternal border. A cardiac echo revealed
  aortic insufficiency and a mobile mass (a
  vegetation) on a leaflet of the aortic valve.
  Blood cultures were obtained three times at six
  hour intervals and all grew the same organism.
  The Gram stain was made directly from that
  culture.
• Gram stain revealed Gram-positive cocci in
  clusters.
• What is the most likely bacterium?

• Answer Streptococcus viridans.
• Subacute Endocarditis.