PA- Microbiology

o Gram staining characteristics
o Morphology
o Metabolic behavior
o DNA sequence

- Unique to bacteria.
- Maintains cell shape.
- Protects the cell from bursting.
- Provides attachment sites.
- Provides a platform for appendages (pili or flagella).
- Differentiates gram positive from gram negative organisms.

• Staphylococcus, Streptococcus, Clostridium, Bacillus

Mycobacterium

Mycoplasma

Rickettsia, Chlamydia

found in all bacteria except Mycoplasma

G(+) bacteria only

G(-) bacteria only

Gives a quick look at the specimen
- Presumptive identification
Can interpret quality of specimen
Can help direct antibiotic therapy
- Based on cell wall composition

- Contain large amount of fatty waxes (mycolic acid) within their cell wall à resist staining by ordinary methods
- Require a special stain for diagnostic à Acid Fast stain.

- Carbolfushin at high concentration,penetrate the lipid cell wall, reach the cytoplasm
- Cytoplasm once stained resist decolorization;acid-alcohol can not dissolve the cell wall and penetrate beneath it hence the name acid fast
- Bacteria that do not have mycolic acid are readily decolorized by the acid-alcohol

acid fast retain the carbolfuchsin dye

not acid fast, decolorized with acid-alcohol , counterstained with methylene blue

Chain = Streptococcus
- Cluster = Staphylococcus

- Chain = Streptobacillus
- Coccobacillus- short bacilli

- Comma-shaped
- Spiral-shaped
- S-shaped

Oral anaerobes
Fusobacterium, Bacteroides, Peptostreptococcus
Streptococci esp. viridans group
Neisseria spp. (incl. meningococcus)
Corynebacterium spp.
Haemophilus spp.

Streptococcus pneumoniae
Haemophilus influenzae
Streptococcus pyogenes (Group A)
Moraxella catarrhalis
Enterobacteriaceae
Yeast

Mycobacterium tuberculosis
Legionella spp.
Brucella spp.

Blood
Cerebrospinal fluid (CSF)
Body fluids (Peritoneal and pleural)

Respiratory tract
Ear, eye and mouth
Skin (wound and abscess)
Urine (mid-stream)
Stool

Sputum samples sent for culture and sensitivity from patients with pneumonia able to expectorate purulent samples and who have not received previous antibiotic therapy. (expectorated)

Specimens that are largely or wholly saliva yield misleading information and should not be sent for culture.

Patient instructed to remove dentures, rinse mouth and gargle with tap water; not with antiseptic mouthwash. Early morning sample preferred.

Fresh, uncontaminated secretions from tracheobronchial tree
Sample must come from windpipe
Reduce contamination of specimen
Rinse mouth with water
Brush teeth before expectorating a specimen
Sputum induction may be needed to obtain acceptable specimen

Induced sputum-collected under respiratory therapist supervision
Patient inhales aerosolized saline (hypertonic saline) until coughing is induced
If sputum induction is not successful
sample with a bronchoscopic lavage
Transtracheal not commonly performed
Patient in a coma with a respiratory infection
Sputum of less than 2ml should not be processed unless obviously purulent

- Used exclusively for isolation of AFB
- Collected from patients unable to produce sputum, particularly young children.
- Gastric lavage must be delivered to the lab immediately so that the acidity can be neutralized.
- Specimen first neutralized and then transported if immediate delivery is not possible.

Sputum samples from tracheobronchial tree more viscous and purulent than saliva
Discard if specimen is mostly saliva
Color, presence of blood, viscosity and odor
Yellow or green sputum occurs in response to an allergic or infectious process
Stringy, mucoid sputum sign of bronchial asthma

thick, green sputum with a unique grapelike/ musty odor

a currant jelly-like sputum

three-layered foul smelling sputum

- Important to evaluate the quality of specimen
- Presence of many squamous epithelial cells in sputum indicates otopharyngeal contamination

Greater than 25 squamous epithelial cells per high power

Less than 10 epithelial cells per low power field

Presence of 25 or more leukocytes with few epithelial cell
Suggest sputum is from lower airway (representative sample)

If leukocytes predominantly eosinophils then the cause of sputum production is allergy
If predominantly PMNs then cause of sputum production is more likely an infection

Determine number of organisms
Organism's morphology, gram stain, location
Capsule
Presumptive diagnosis

(most common cause of bacterial pneumoniae) is an encapsulated lancet-shaped gram-positive diplococcus

appears as an encapsulated gram-negative rod

appears as a gram-negative coccobacilli

appears as gram-positive cocci in clusters