HCS228 Culture-Based Diagnostic & Typing Methods 2024-25 PDF

Culture-based Diagnostic & Typing Methods Dr Lewis Bingle HCS228 (2024-25)  Lecture Summary Introduction to diagnostic microbiology Microscopy Culture Biochemical tests 2 What is Diagnostic Microbiology?...

Culture-based Diagnostic & Typing Methods Dr Lewis Bingle HCS228 (2024-25)  Lecture Summary Introduction to diagnostic microbiology Microscopy Culture Biochemical tests 2 What is Diagnostic Microbiology? Are there any pathogens in this clinical sample? What are they? What disease features might we expect? What antibiotics can we use to treat? 3  What is Diagnostic Microbiology? Identification of potential pathogens – e.g. to species level Typing – Distinguishes between strains within species Antimicrobial (antibiotic) susceptibility testing Pathogenicity profiling Focus on clinical applications, but also common ground with… – Veterinary microbiology – Plant (crop) pathology – Food microbiology 4  Current Diagnostic & Typing Methods Specimen type/clinical details of patient/antibiotic therapy Macroscopic observation of sample Microscopy +/- stains Tolerance to environmental conditions Colonial morphology Biochemical testing Susceptibility to antibiotics and chemical inhibitors Requirement for growth factors Host (patient) biomarkers (e.g. CRP) Serological Molecular Immunological Phage typing 5  Macroscopic Investigations Type of specimen – is it appropriate? Volume of specimen – is it enough? Typical appearance of an infected specimen – cloudy, coloured etc..? Blood present? Parasites visible (faecal sample)? 6  Microscopy Direct microscopy of specimens: – detect bacterial cells, fungal elements, parasites, inclusions in cells Microscopy of slides prepared from cultures Many different types of microscopy – Bright-field – Phase contrast – Fluorescence – Electron Many different stains & labels to highlight pathogens 7  Optics Microscopy is covered in detail in HCS227 Visualising bacteria will normally require use of a 100x oil immersion objective (1000x overall magnification) without a coverslip Fungi and protozoan pathogens are larger and may be visible using a 40x objective lens (400x overall magnification) Phase contrast or fluorescence microscopy require specialised optics 8  Microscopy & Staining Bacteria Gram staining (1° dye = crystal violet) Acid-fast bacteria e.g. Mycobacterium (TB): Ziehl-Neelsen staining (1° dye = carbol fuchsin) See UK Standards for Microbiology Investigations TP39: Staining procedures 9 H. C. Gram  Gram Stain Chemistry Crystal violet dissociates into + & - ions that penetrate envelope of both Gram-positive & Gram-negative cells CV+ ion interacts with negatively charged components of bacterial cells (e.g. nucleic acids) staining cells purple Iodine (I-) mordant* interacts with CV+ to form large CVI complexes in cell At this point: Gram-positive and Gram-negative cell are stained purple 11 *Mordant: substance that combines with a stain & thereby fixes it in stained material.  Gram Stain Chemistry Decolorizing agent (ethanol / acetone) interacts with membrane lipids & cell wall OM of Gram-negative cell lost, exposing thin peptidoglycan (PG) layer - cell wall becomes leaky & allows large CVI complexes to be washed from cell Highly cross-linked & multi-layered PG of Gram-positive cell is dehydrated by ethanol & traps large CVI complexes within cell After decolourization, Gram-positive cell remains purple in colour, whereas Gram-negative cell loses purple colour Gram-negative cells revealed by counterstain e.g. +ve charged dye safranin* Final result: Gram-positive cell purple; Gram-negative cell pink to red 12 *Other counterstains are available e.g. carbol (basic) fuchsin  Notes on Gram Staining Caution: bacterial cell structure or culture conditions may cause abnormal Gram-staining! Culture age may influence results of Gram stain “Gram-variable” staining (mix of pink & purple cells) can be due to poor staining technique or cell biology Some bacteria do not stain as expected e.g. genus Acinetobacter are Gram-negative cocci resistant to decolorization step  often appear Gram-positive For Mycobacterium spp. (e.g. M. tuberculosis), waxy cell envelope prevents Gram staining Misinterpretation of Gram stain results can lead to misdiagnosis or delayed diagnosis of infectious disease 13  Microscopy & Staining Fungi Direct examination: potassium hydroxide (KOH) procedure (fungi have chitinous cell walls and are resistant to KOH) Calcofluor White (CFW) non-specifically binds chitin & cellulose in cell wall & fluoresces bright green-blue Lactophenol blue preparation A picture containing hydrozoan Description automatically generated 14  Microscopy & Staining Protozoa Direct microscopy of unfixed sample e.g. Entamoeba histolytica (amoebic dysentry); Trypanosoma brucei in blood – see image below and video here: https://www.youtube.com/watch?v=txMrdLbyFLM&list=PLId6 rTkhcvZXWfV7CFljd7-Ut4U0TkNpH&index=1 Trichrome, Giemsa stains 15  Microscopy: Pros & Cons Advantages Speed (PoC potential?) Direct from specimens and or indirect from cultures Limited technology required with most methods Disadvantages Requires skilled “hands on time” Subjective (operator dependent) Lacks sensitivity (needle in a haystack) Affected by specimen quality Specificity may be limited 16  Culture Choice of media What are the likely pathogens? What is the specimen type? Choice of culture conditions Bacteria normally 37 C / 35 C Fungi normally 30 C CO2 Anaerobic cabinet / jar See Ford Medical microbiology Ch. 3 17  Culture Media Media types: Non-selective Supplemented e.g. chocolate agar Differential e.g. blood agar (haemolysis) Selective e.g. antimicrobial agar Chromogenic See e.g. http://www.eolabs.com/product-category/ppp/ppm- clinical-veterinary-2/ to get an idea of range Also: Cell culture (i.e. virus or obligate intracellular bacteria can be cultured on human or animal cell lines) 18  Non-selective media for non-fastidious microbes Defined / minimal media – e.g. M9 minimal (mostly for molecular biology / genetics research) Undefined (rich) media – e.g. nutrient agar, LB agar, tryptone soy broth Diagnostic information: – Macroscopic (colonial) morphology – Smell (note health & safety issues!) “Anything grows” (see environmental contamination lab) 19  Supplemented media for fastidious microbes BHI broth Blood (e.g. horse blood) Chocolate agars (lysed RBCs e.g. for CSF) Growth factor X+V discs for identification of Haemophilus influenzae Charcoal additive (adsorbs toxic metabolites) 20  Selective media For specimens from non-sterile sites Supplemented with antimicrobials / other inhibitors to restrict growth of commensals & allow growth of pathogens Example 1 (Ford, 2014) Leg ulcer swab on blood agar: aztreonam (RHS) inhibits Gram- negative but not Gram-positive growth & “unmasks” S. aureus 21  Selective media Example 2 (Ford, 2014) Optochin disc differentiates optochin-sensitive Streptococcus pneumoniae from resistant α-haemolytic (green haemolysis) streptococci (e.g. S. viridans) 2 22  Differential media Highlight distinctive metabolic activities Carbohydrate source utilisation → acid → pH change Medium contains pH indicator & carbohydrate source e.g. CLED: lactose, bromothymol blue; MacConkey: lactose, neutral red; DCA; MSA (also selective due to 7.5 % NaCl); XLD agars MSA e.g. wound swab CLED MacConkey S. aureus S. epidermidis Lac+ Lac- 23  Chromogenic Media Chromogenic enzyme substrates are hydrolysed in the presence of specific microbial enzymes e.g. glycosidases High specificity Substrates used are often based on derivatized indoxyl (related to indigo dye) e.g. E. coli has diagnostic enzyme ß-glucuronidase & can be identified using substate indoxyl-ß- glucuronide enzyme Chromogen Chromogen Off Linker On Linker cleavage substrate products 24  There’s something growing on the plate! Now what? Consider your data….. What were you trying / expecting to grow? What does the medium contain (e.g. selective agents)? Under what atmospheric conditions did the pathogen grow (aerobic / CO2 supplementation / anaerobic)? At what temperature did the pathogen grow? What does it look like (colony morphology)? What further tests can you perform to confirm or extend provisional identification? What if nothing grows? Can be common occurrence for some sample types Inappropriate choice of culture conditions? Non-infectious cause? 25  Colonial morphology Organism appearance on an agar-based culture media Colour Shape Translucency Outline Changes brought about in the medium (e.g. haemolysis of blood agar) 26 What does the colony look like……? Effects on medium Mucoid Size & shape Coloured 27 What does the colony look like……?  Tolerance to Environmental conditions (covered at level 1) Temperature Atmosphere Psychrophile Obligate aerobe Thermophile Obligate anaerobe Mesophile Facultative anaerobe Aerotolerant anaerobe Microaerophilic Capnophilic (high [CO2] 29  Biochemical Testing Examples include Amino acid metabolism Carbohydrate oxidation/fermentation Utilisation of a carbon source Nitrate reductase Catalase Oxidase Coagulase Hydrolases DNase etc., etc., etc. 30  Biochemical Testing Identification of organisms previously isolated in pure culture Individual biochemical tests differentiate between 2 similar organisms Example Tryptone broth cultures after addition of Kovács reagent (see Enterobacter Escherichia image) aerogenes coli Escherichia & Enterobacter closely related (Order Enterobacterales) E. coli makes indole Enterobacter does not 31  Biochemical Profiling Biochemical test panel / strips e.g. API (bioMérieux, 1970) – Panel of miniaturized biochemical tests – 7-digit code (Analytical Profile Index) – A “Gold Standard” method but can be slow & imprecise Automated systems e.g. Vitek 32  The “Great Plate Count Anomaly” Plate counts from environmental samples are much (orders of magnitude) lower than direct counts Possible reasons include: – Differing nutritional requirements (fastidiousness) – Non-cultivatable dormant states – Microbes need communities Can this apply to clinical microbiology? – Whipple’s disease Figure: Evolution (2007) CSHL Press 33  Problems With Culture-Based Methods Problems with obtaining organisms in pure culture and identifying them by traditional phenotypic approaches Labour and time-intensive Error-prone (e.g. fails to discriminate between closely related species) Insensitive (common bacteria swamp rare ones) Many species resist in-vitro culture Plate counts from some samples can be lower than direct counts of bacteria One solution is the use of modern molecular methods that avoid need for culture 34  Modern methods Immunological (detecting antigens) / Serological (detecting antibodies) – ELISA Molecular – PCR / NAATs – MALDI-TOF Sequence-based methods – Whole-genome DNA sequencing 35 36  Learning Outcome You should be able to explain how we can use microscopy & culture-based methods (including biochemical testing) to identify clinically important bacteria 37