Skip to content

Add your earlier progress to your account?

From before you signed in, this browser has:

Which plan do you want to keep?

You answered the plan questions before you signed in, and those answers don't match the plan saved on your account.

SettingSaved planNew answers

Study progress is waiting to be saved

Direct stains, media and incubation

16 min

Skills you'll build3 skills
  • Read a direct Gram stain and compare it with the culture growth
  • Select primary media for the specimen source and suspected organism group
  • Choose ambient air, CO₂, or anaerobic incubation for each culture plate

At the bench: Select specimen preparation, media, and incubation for a culture · Read an organism stain and communicate a significant preliminary finding

Try first

Try first

A cytocentrifuged Gram stain of pleural fluid shows a few neutrophils and no organisms. What does the smear tell you?

Keep going to see the reasoning.

Right. Keep going to follow the reasoning.

Keep going to see the reasoning.

Keep going to see the reasoning.

Get the idea

Use the explanation to check your first decision.

Reading the direct Gram stain

The direct smear is the first look at the specimen. Spread the most purulent part, fix it with methanol, and cytocentrifuge sterile fluids first, which raises the number of visible organisms up to about 100-fold.1,2 Scan at low power, then read bacteria under oil at 1,000×. About 10⁵ organisms per mL are needed for one per oil field, so a negative smear does not exclude an infection with few organisms.1

Describe the shape, arrangement and Gram reaction as precisely as the smear allows. Count white cells and squamous epithelial cells, and note organisms inside cells.1

Stain a slide at the Gram stain bench

Comparing the smear with the culture

Every organism type on the smear should appear in the culture.1

  • Organisms seen that fail to grow suggest a missing medium or atmosphere, such as Haemophilus with no chocolate agar. A misread smear is considered last.
  • Heavy growth that the smear missed suggests an organism load below the smear's detection limit, a different part of the specimen, or contamination of the culture.

Media and atmosphere

MediumPurpose
Sheep blood agarGrows most routine organisms and shows hemolysis
Chocolate agarSupplies hemin (X factor) and NAD (V factor) for Haemophilus and other fastidious organisms
CNA or PEA agarSuppresses gram-negative rods
MacConkey agarSuppresses gram-positive organisms and shows lactose fermentation
Thioglycolate brothRecovers small numbers of organisms, including many anaerobes

Inoculate plates before broth, and less selective media before more selective ones.1

Routine cultures incubate at 35 ± 2 °C and are read at 18 to 24 hours. Blood and chocolate plates go into 5% to 10% CO₂, which N. gonorrhoeae, H. influenzae and S. pneumoniae need or grow better in.1,3 MacConkey and other enteric plates incubate in ambient air, because CO₂ acidifies them and distorts their color reactions.1,4 Anaerobic plates go into a jar, pouch or chamber, are first opened at 48 hours and are kept at least 5 days.1

Follow the bench demonstration

Sources for this step
References
  1. Tille PM. Bailey & Scott's Diagnostic Microbiology. 15th ed. Elsevier; 2021.
  2. Tunkel AR, Hartman BJ, Kaplan SL, et al. Practice guidelines for the management of bacterial meningitis. Clin Infect Dis. 2004;39(9):1267-1284. doi:10.1086/425368
  3. Papp JR, Schachter J, Gaydos CA, Van Der Pol B. Recommendations for the laboratory-based detection of Chlamydia trachomatis and Neisseria gonorrhoeae—2014. MMWR Recomm Rep. 2014;63(RR-2):1-19. Accessed September 27, 2026. https://www.cdc.gov/mmwr/preview/mmwrhtml/rr6302a1.htm
  4. Hasan MR, Suleiman M, Ilagan E, et al. Growth of clinically important gram-negative bacteria on MacConkey agar under aerobic versus CO₂-enriched environment. J Clin Microbiol. 2019;57(12):e01441-19. doi:10.1128/JCM.01441-19

Watch one

Read each decision and its explanation.

An abdominal abscess aspirate arrives in anaerobic transport for aerobic and anaerobic culture. The laboratory set up blood and chocolate plates in CO₂, a MacConkey plate in ambient air, anaerobic plates in a jar and a thioglycolate broth.

  • Gram stain: many neutrophils, many gram-negative rods of varied shape and length, many gram-positive cocci in chains, and a few large gram-positive rods.
  • Aerobic plates at 24 hours: moderate growth of one lactose-fermenting gram-negative rod.
  • Anaerobic jar: not yet opened.

How do the smear and the 24-hour culture compare?

  1. Check the cells first: many neutrophils, so the aspirate is an adequate specimen.

    Neutrophils show that the specimen came from the site of inflammation.

  2. List the morphotypes: gram-negative rods, gram-positive cocci in chains and large gram-positive rods, all numerous.

    Each organism type on the smear should be accounted for in the culture.

  3. Compare the aerobic growth: the lactose fermenter accounts for some gram-negative rods. The cocci and the large rods are not accounted for.

    Accounting for one morphotype leaves the others unexplained.

  4. Check what is still incubating: the anaerobic plates have not been read.

    Anaerobic plates are first opened at 48 hours, because early exposure to oxygen reduces recovery.

  5. Report the smear as seen, report the aerobic organism as a preliminary result, and state that the anaerobic culture is to follow.

    A final report now would account for only part of what the smear shows.

The smear shows more than the 24-hour aerobic culture, and the unmatched organisms fit anaerobes still growing. Report the aerobic growth as preliminary. The final report waits for the anaerobic plates, first read at 48 hours and kept at least 5 days.

Practice this skill

Your turn

Problem 1 of 3 · Hints available

A sputum Gram stain shows many small gram-negative coccobacilli with neutrophils. Only a sheep blood agar plate was inoculated, and it grows normal oral flora. What does the comparison suggest?

Incorrect. Many organisms seen on a smear do not grow on sheep blood agar, so failure to grow first suggests a missing medium. A misread smear is considered only after the culture setup is checked.

Incorrect. Contamination adds growth that the smear lacks, so it cannot explain a smear showing organisms that failed to grow.

Correct. Haemophilus needs free NAD (V factor), and an enzyme in sheep erythrocytes inactivates it. Chocolate agar releases both X and V factors from heat-lysed blood. A chocolate plate is added on the next specimen.

Hint
  1. Organisms seen on the smear were viable in the specimen. Consider what they need to grow.
  2. Consider which medium supplies those growth factors.

Review Direct examination

Problem 2 of 3 · Hints available

A laboratory professional places a stool culture's MacConkey, xylose-lysine-deoxycholate (XLD), and Hektoen enteric plates in the 5% CO₂ incubator with the blood and chocolate plates. What is the problem?

Incorrect. The organisms do grow. These plates are read by color, and added CO2 acidifies the medium and distorts the pH-indicator reactions.

Correct. MacConkey, XLD, and Hektoen enteric agar are incubated at 35 ± 2 °C in ambient air, because added CO2 shifts the indicator reactions the differentiation depends on and can reduce recovery of some gram-negative bacilli.

Incorrect. Microaerophilic incubation at 41 to 42.5 °C is the setup for Campylobacter selective agar. MacConkey, XLD, and Hektoen enteric agar are read after incubation in ambient air.

Hint
  1. Consider how MacConkey, XLD and Hektoen plates show their reactions.
  2. Consider the effect of CO₂ dissolved in the medium.

Review Incubation

Problem 3 of 3 · On your own

Synovial fluid from a swollen knee arrives for bacterial culture. Which primary setup is correct?

These plates select for enteric pathogens in stool. A joint is a normally sterile site, and these media would suppress many of the organisms expected there.

Sheep blood agar alone cannot grow Haemophilus, and fastidious organisms such as Neisseria do better with chocolate agar. A sterile-site setup includes it.

Left chocolate agar off a respiratory setup

An enzyme in sheep red cells inactivates NAD, so Haemophilus fails to grow on sheep blood agar alone. Chocolate agar supplies X and V factors released from heat-lysed red cells. A respiratory or sterile-site setup without it can grow only normal flora from a specimen whose smear shows the pathogen.

Blood agar grows most organisms, chocolate agar adds the X and V factors fastidious organisms need, and the broth recovers organisms present in small numbers.

Two selective plates provide no medium for fastidious organisms. A normally sterile fluid needs enriched, nonselective media.

Review Media selection and inoculation

Use it

Read the case evidence, then make each decision. The step is complete without it.

  • Jonah Maxwell, 4, has red, crusted eyes with a thick discharge.
  • His pediatrician sends a conjunctival swab for bacterial culture and a direct smear on a slide.
  • The Gram stain shows many neutrophils and many small gram-negative coccobacilli.
Decision 1 of 3

Which plates do you set up?

Sheep red cells contain an enzyme that inactivates NAD. The coccobacilli on the smear may fail to grow on blood agar alone.

Left chocolate agar off a respiratory setup

An enzyme in sheep red cells inactivates NAD, so Haemophilus fails to grow on sheep blood agar alone. Chocolate agar supplies X and V factors released from heat-lysed red cells. A respiratory or sterile-site setup without it can grow only normal flora from a specimen whose smear shows the pathogen.

Chocolate agar supplies the X and V factors that Haemophilus needs, and blood agar grows S. pneumoniae and staphylococci. Both are needed for a child's conjunctivitis.

Each of these plates suppresses part of the flora, and neither supplies V factor. The organisms seen on the smear need enriched media.

Review Media selection and inoculation

Decision 2 of 3

Where do the plates incubate?

H. influenzae and S. pneumoniae need CO₂ or grow better in it. The enriched plates go into the CO₂ incubator.

Ambient air is used for enteric plates. The organisms expected here grow poorly without added CO₂.

Anaerobes are not expected on the conjunctiva, and the organisms suspected here grow in air with added CO₂. Waiting 48 hours delays the result.

Review Incubation

Decision 3 of 3

How do you report the Gram stain?

The report describes the cells, the shape and the Gram reaction as precisely as the smear allows. The culture identifies the organism.

A Gram stain shows shape and staining. The species needs growth-factor tests or another identification method.

This omits the shape and the neutrophils. The full description gives the clinician more information on the same day.

Review Direct examination

The setup follows from the small gram-negative coccobacilli on the smear. They suggest Haemophilus, which needs chocolate agar and CO₂. At 24 hours, small gray colonies grew on chocolate agar and nothing grew on blood agar.

For reference

Sources checked