anaerobic growth

Anaerobic Growth Media & Methods

The cultivation of anaerobic bacteria poses a special problem because anaerobes might be killed by exposure to oxygen, so special reducing media must be used, e.g. sodium thioglycolate.

Special Treatment of Anaerobic Media

The medium is heated shortly before use to drive off absorbed oxygen, then stored in tightly capped tubes.

Anaerobic Growth Methods

1. Candle jar technique; 2. Anaerobic chamber

1. Candle Jar Technique

Plates/tubes are placed in a jar with a lighted candle inside; the jar is capped. The candle extinguishes when the atmosphere reaches approximately 10% CO₂.

2. Anaerobic Chamber

Oxygen is removed via a packet of chemicals (sodium bicarbonate + sodium borohydride), moistened with water, and the jar is sealed. Hydrogen and carbon dioxide are produced by the reaction. It is a transparent enclosure filled with inert, oxygen-free gas; organisms/materials enter and leave through an air lock, and manipulations are done via glove ports.

Candle Jar vs Anaerobic Chamber

Candle jar = quick/simple and NOT true anaerobic because some O₂ remains; anaerobic chamber = true, strict anaerobiosis.

Colony

A visible mass of microorganisms all originating from a single mother cell.

Colony Morphology

The visible characteristics of a colony; an important aspect of identification, although NOT reliable alone because many bacteria share similar colony morphology.

Growth on Solid Media — Agar Plate

Colony characteristics include: 1. Size; 2. Form/Shape; 3. Elevation; 4. Margin/Edge; 5. Surface; 6. Consistency/Texture; 7. Colony Density; 8. Odor; 9. Amount/Abundance; 10. Chromogenesis/Pigmentation.

1. Size

Diameter in mm, or described as pinpoint/small/medium/large. Colonies >5 mm are likely motile. Gram(+) bacteria usually have smaller colonies than Gram(−) bacteria.

2. Form/Shape

The shape of the colony.

3. Elevation

The side view of the colony; tilt the plate to observe.

4. Margin/Edge

Irregular shape/margin may indicate motility, e.g. Proteus swarming. S. pneumoniae: smooth with capsule vs rough without capsule. B. subtilis: wavy/lobed margins due to flagella.

5. Surface

May be smooth, glistening, rough, dull, rugose (wrinkled), etc.

6. Consistency/Texture

Determined by touching with a sterile loop; may be dry, moist, viscid, brittle/friable, or mucoid.

7. Colony Density

May be transparent, opaque, translucent, or iridescent; transillumination can be used.

8. Odor

May include ammonia (NH₃), rotten egg (H₂S), alcoholic, fecal, etc.; may be absent/present, putrid, fruity, or rancid.

9. Amount/Abundance

None, slight, moderate, or large.

10. Chromogenesis/Pigmentation

Pigments may be intracellular or extracellular soluble pigments. Examples: green = P. aeruginosa; buff = M. tuberculosis in L-J; red = Serratia marcescens.

Order for Describing Colony

SIZE → FORM → ELEVATION → MARGIN → SURFACE → CONSISTENCY → DENSITY → ODOR → AMOUNT → PIGMENT

Growth on Solid Media — Agar Slant

1. Filiform; 2. Echinulate; 3. Beaded; 4. Effuse; 5. Arborescent; 6. Rhizoid

Filiform

Continuous, threadlike, smooth edges.

Echinulate

Continuous, threadlike, irregular edges.

Beaded

Non-confluent to semi-confluent colonies.

Effuse

Thin, spreading growth.

Arborescent

Tree-like growth.

Rhizoid

Root-like growth.

Growth on Liquid Media

1. Opacity; 2. Surface Growth; 3. Subsurface Growth/Deposits

Opacity — Liquid Media

Opaque = no light; Translucent = partial light; Transparent = full light.

Surface Growth — Liquid Media

Example: pellicle formation.

Subsurface Growth/Deposits — Liquid Media

Sediment below the surface or at the bottom.

Inoculation of Culture Media — Streak Plate Methods

The goal is obtaining isolation of individual bacterial species from a sample, which is the first step in the identification process.

Streak Patterns

Range from simple to complex; they separate cells on the agar surface so isolated colonies can grow.

Streak Pattern According to Cell Density

Quadrant streak = high cell density; zigzag = lower cell densities.

Quadrant Streak Method

Usually performed as the initial streak for isolation of two or more bacterial species in a mixed culture with suspected high cell density.

Quadrant Streak Method — 4 Steps

1. Obtain sample with sterile loop. Lift lid as a shield. Streak back and forth at the plate's edge (Streak I). Do not cut the agar. Replace lid. 2. Flame the loop base-to-tip, let cool. Rotate plate approximately 90°, touch an uninoculated region to cool further, then streak again (Streak II), starting at one end of Streak I. Flame the loop. 3. Sterilize/cool the loop again, rotate approximately 90°, and make the third streak (Streak III) beginning in Streak II. Flame again. 4. Sterilize/cool the loop, make the fourth streak (Streak IV) beginning in Streak III and extending to the plate center. Do not enter any streak except the third. Label plate and incubate inverted.

Step 1 — Quadrant Streak

Obtain sample with sterile loop. Lift lid as a shield. Streak back and forth at the plate's edge (Streak I). Do not cut the agar. Replace lid.

Step 2 — Quadrant Streak

Flame the loop base-to-tip and let it cool. Rotate plate approximately 90°, touch an uninoculated region to cool further, and streak again (Streak II), starting at one end of Streak I. Flame the loop.

Step 3 — Quadrant Streak

Sterilize/cool the loop again, rotate approximately 90°, and make the third streak (Streak III) beginning in Streak II. Flame again.

Step 4 — Quadrant Streak

Sterilize/cool the loop, make the fourth streak (Streak IV) beginning in Streak III and extending to the plate center. Do not enter any streak except the third. Label plate and incubate inverted.

Zigzag Method

Use a cotton swab to streak the agar surface in a zigzag pattern to get isolated colonies after incubation. Be careful not to cut the agar. Dispose of the swab properly in a biohazard container.

Quadrant Streak Memory Hack

Quadrant streak = 4 progressively-diluting streaks rotated approximately 90° each time, always cooling/flaming the loop between streaks and only touching the previous streak. This takes a heavily mixed sample toward single isolated colonies.