Lab manual^*

Lab micro order forms: provided by the lab

Marking pen for labeling samples

Ophthalmic anesthetic (proparacaine preferred)

Microscope slides

Microscope^†

Sterile dacron swabs

Methyl alcohol

Distilled water source

Kimura spatula

Alcohol lamp or propane burner

Bacterial/viral culturettes

Culture media

Blood agar

Chocolate agar

Commercial chlamydia transport kits (nonrefrigerated type) Example: Gen-Probe (Pace) for conjunctival and urethral specimens

Gram stain materials

Diff-Quick or Hemacolor stain kits

^* By Haesert S, Clinical Manual of Ocular Microbiology and Cytology. St. Louis, MO: Mosby, 1993, is highly recommended by this author but out of print.

^† Only needed if you do your own stains.

I.

GRAM-POSITIVE ORGANISMS

A.

Staphylococci

1.

S. aureus

2.

S. epidermidis

B.

Streptococci

1.

α-Hemolytic streptococcus

2.

β-Hemolytic streptococcus

3.

Streptococcus pneumoniae

C.

Bacilli (rods)

1.

Bacillus

a. B. anthracis

b. B. cereus

c. B. subtilis

2.

Corynebacterium

a. C. diphtheriae

b. C. xerosis

c. C. pseudodiphtheriticum

3.

Listeria sp.

4.

Nocardia sp.

5.

Mycobacterium sp.

II.

GRAM-NEGATIVE ORGANISMS

A.

Neisseria

1.

N. gonorrhoeae

2.

N. meningitidis

B.

Bacilli

1.

Enterobacteriaceae

a. Escherichia coli

b. Shigella sonnei and Shigella flexneri

c. Klebsiella pneumoniae and Klebsiella oxytoca

d. Serratia marcescens and Serratia liquefaciens

e. Proteus mirabilis and Proteus vulgaris

2.

Moraxella

a. M. lacunata

b. M. nonliquefaciens

c. M. bovis

3.

Haemophilus

a. H. influenzae

b. H. aegyptius (Koch-Weeks bacillus)

4.

Brucella

a. B. abortus

b. B. suis

c. B. melitensis

5.

Pseudomonas

a. P. aeruginosa

b. P. cepacia

CA-MRSA

HA-MRSA

Name

Community-associated methicillin-resistant S. aureus

Health care-associated methicillin-resistant S. aureus

Risk factors

Otherwise healthy people sharing personal items such as bar soap, towels, razors, athletic equipment, direct skin contact with lesion

Recent hospitalization or medical procedures on immunocompromised, postsurgical, dialysis, or long-term care patients

Body site

Clusters of skin or soft tissue boils, “pimples,” abscesses

Septicemia, pneumonia, wound or postsurgical sites

Incidence^*

14% but increasing

86%

Lab reports

Gram + cocci catalase and coagulase +

Gram + cocci catalase and coagulase +

Antibiotic sensitivity

Variable, C & S required, multiresistant

Variable, C & S required, multiresistant

Outcome

Typically somewhat better than HA

Can be poor, lengthy course

Prevention

Frequent handwashing, hand sanitizer, cover wound, patient education

Frequent handwashing, cover wound, universal precautions, patient education

CDC Web site www.cdc.gov.

^* Klevens et al., JAMA 2007;298:1763-1771.

Characteristic

Staphylococcus

Streptococcus

Haemophilus

Morphology

Cocci in clusters

Cocci in pairs and chains

Pleomorphic rods

Gram stain

Positive

Positive

Negative

Pathogenesis

Exotoxins, enzymes

Tissue invasion and multiplication, enzymes

Unknown

Exudate

Suppurative

Serous

Suppurative

Ocular presentation

Blepharitis, dysfunction of glands

Blepharitis, cellulitis

Blepharitis, cellulitis

Antibiotic sensitivity

Bacitracin^*, moxifloxacin^*, gatifloxacin^*, gentamicin^*, cephalosporins, erythromycin

Penicillin^*, moxifloxacin^*, gatifloxacin^*, many strains now resistant to erythromycin

Penicillin^*, polymyxin B*, sulfonamides, erythromycin

Antibiotic

Wide variation, sulfonamides <15%

Aminoglycosides

^* Antibiotics of choice.

Inflamed conjunctivae

Normal

Organisms

No. of patients

% of total

No. of patients

% of total

ANAEROBES

Peptococcus

7

5.4

2

3.2

Peptostreptococcus

10

8

Propionibacterium

30

22.9

6

10.0

Bacteroides melaninogenicus

3

2.3

Bacillus fragilis

1

0.76

Actinomyces

1

0.76

Bifidobacterium

1

0.76

Fusobacterium

1

0.76

AEROBES

S. epidermidis

65

49.6

25

42

S. aureus

23

17.5

2

3.3

Streptococcus viridans

17

13.0

5

8.3

Group D Streptococcus

4

3.0

5

8.3

β-Hemolytic Streptococcus Group A

3

2.3

S. pneumoniae

4

3.0

Lactobacillus

5

3.8

Diphtheroids

24

18.3

15

25.0

H. influenzae

9

6.9

Neisseria sp.

9

6.9

3

5

Neisseria meningitidis

2

1.5

Acinetobacter

1

0.76

1

1.6

K. pneumoniae

1

0.76

1

1.6

Pseudomonas sp.

1

0.76

2

3.3

E. coli

2

1.5

Candida

1

0.76

3

5.0

Heat fix or fix smear in methanol for 5 min and then allow to air-dry.

Flood with crystal gentian violet for 1 min.

Rinse with tap water.

Flood with Gram’s iodine for 1 min.

Rinse with tap water.

Decolorize with acid alcohol until solvent flows clearly from the slide (10 to 20 s).

Rinse with tap water.

Counterstain with safranin, flood for 1 min.

Rinse with tap water.

Air-dry or blot gently.

View on 100× oil immersion.

It is preferable to collect and fix the Gram stain specimen and send it to the lab for staining and evaluation. An unstained Gram stain prep should be sent with every culture. Staining and evaluating Gram stain preps in-office require a higher level of CLIA certification than performing Diff-Quick or Hemacolor specimens in-office.

Name of stain

Usage

Results

Procedure

Periodic acid-Schiff (PAS)

Visualize goblet cells to detect mucin deficiency

Goblets stain magenta/pink Fungi hyphae also stain pink

Collect by impression cytology, place in ethanol, and send to the lab

Acid fast (Ziehl-Nelsen, Kinyoun)

Identifying mycobacteria

Organisms stain red if acid fast

Collect sputum or scraping and send to the lab

Wright, Giemsa

Differentiate white blood cell types

Classic staining of lymphs, eosinophils, neutrophils, monocytes

Collect ocular scraping or blood, fix, and send to the lab

Diff-Quick, Hemacolor

Rapid in-office modification of Wright and Giemsa

Same as for Wright or Giemsa

Collect scraping or blood on slide, five 1-s swirls in fixative, then soln 1, then soln 2, rinse, and view on 10× and 40×

Gram

Identify bacterial shape and Gram reaction

Purple/blue Gram + Pink/red Gram –

Collect scraping, heat or methanol fix, and send to the lab

Papanicolaou (PAP)

Detection of malignant cells, intranuclear and intracytoplasmic cellular inclusions

Transparency allows visualization of chlamydial, cytomegalovirus (CMV), and herpetic inclusion bodies

Collect scraping, fix, and send to the lab