Microscopy

Tereza Kopecká

Remember!

• Microbiologists love microscopy.

• We perform it always, if possible and informative.

• If it‘s possible to identify through microscopyalone, we take it. (typically parasitology and mycology)

What do we need for microscopy?

• A microscope – let‘s focus on the light one.

• Slides– Native (with saline solution or KOH)

– stained• Gram stain – bacteria, yeasts

• Ziehl-Neelsena – mycobacteria, nocardiae, cryptosporidia

• Giemsa – pneumocysts, parasites

• Lactophenol blue - molds

• India ink (Burri stain) – capsules – Cryptococcus

What do we observe?

• New samples

• Cultures

• Positive blood cultures

– Blood sample from a septic patient

– Blood culture bottle

– Incubation and detection of chemical changesinside

– BD Bactec or BactALERT signalizes positive

New samples

• The liquid ones. The only swab is the MOP

– Sputum and other respiratory

– CSF

– Pus, liquid contents of cavities, etc.

• Bacteriologists never look at stools but

– Parasitologists do (+tape!)

– Virologists as well (electron microscope)

Why observe cultures?

• It might help with identification

– If we have MALDI-TOF, no need

– Not every lab has a MALDI, for chemicalidentification, basic morphology is needed

• In molds, it‘s absolutely necessary

Why observe positive blood cultures?

• We reassure ourselves about real positivity– Do you see no bacteria? Something is wrong

– Let‘s check the curves at Bactec

• We obtain basic data about the microbes– Immediate treatment

– An impulse for other identification methods• Sprout hyphae in yeasts – 2 hours

– A correct susceptibility testing• MH agar with horse blood in cocci forming chains

• Correct sets of antibiotics

Sputum microscopy

• We are interested in:– Human cells

– microbes

• Gram stain is the basic one

• Human cells show us:– Sample validity (Bartlett score)

• With more leukocytes and less squamous cells, thesputum is better

– Intensity of the inflammation• The appearance is quite different in abscess and mild

Sputum microscopy

• We can see the agents!

– Lancet-like G+ diplococci

– G- cocci in tetraplets

– Tiny G- coccobacilli

Sputum microscopy for TB

• Can be informative but must be targeted

– Different staining method (Ziehl-Neelsen)

– Lower sensitivity

• Negative slide does not mean a healthy patient

CSF microscopy

• Routine or suspect neuroinfection

• Quantity of leukocytes and the appearance ofthe microbes must be reported to thephysician

• If they are not present, it‘s an impulse forother methods like PCR or serology

• CSF microscopy might be a life-savingprocedure

A special topic: sprout hyphae

• Yeasts put in horse serum for 2 hours

• If it‘s Candida albicans, broad hyphae are formed

• Mycology practicals…

Let‘s go!