# Microcalorimetry--Versatile Method of Describing Bacterial Growth
## 2 Materials and Methods  
### Classic Method of Infections Diagnosis  
#### 2.1.1 Isolation and Identification of Bacterial Strains  
For this study, 3 infected implants were used, extracted from hospitalized patients in the orthopedics and traumatology department of SUUB. Sampling was performed with a sterile swab impregnated with SSF; the entire surface of the prosthesis was wiped using this swab. The SSF suspension obtained was centrifuged (4000 rpm, 10 min); the sediment obtained, as well as the tissue collected from the samples, were kept at \(-70\)\({}^{\circ}\)C. The swab was discharged on Petri dishes with blood agar and with Sabouraud agar, but also in liquid medium--simple broth. Incubation was performed for 24 h at 37 \({}^{\circ}\)C (and at 28 \({}^{\circ}\)C for any fungi on the Sabouraud agar plate). After 24 h, a blood agar plate and a Sabouraud plate were seeded from the broth, the plates being incubated under the same conditions mentioned above. 1.2 Strain Susceptibility Testing to Antibiotics--Kirby-Bauer Disk Diffusion Susceptibility Test Protocol  
Following the recommendations of the 2018 edition, CLSI 2018, the bacterial suspensions made to obtain a 0.5 McFarland index were inoculated into the cloth on Muller Hinton (MH) agar test medium with the sterile swab. Disks inoculated with different antibiotic concentrations recommended by the standard procedures were applied to the inoculated plates. After incubation at 37 \({}^{\circ}\)C for 24 h, the results were determined by measuring the diameter of the zones of inhibition. The antibiotics tested were beta-lactam antibiotics: penicillin (P), cefoxitin (FOX), ceftroline fosamil (CPT); aminoglycos: gentamicin (CN); macrolides: azithromycin (AZM), erythromycincemic (E); tetradcyclines: tetracycline (TE); fluoroquinolones: ciprofloxacin (CIP); limcosamides: clindamycin (DA); folic acid antagonists: trimethoprim sulfamethoxazole (SXT); ansamycins: rifampicin (RA); and oxazolidine: linezolid (LZD). The reference strain used as a control was S. aureus strain (ATCC 25923). 2.1.3. Testing the Virulence Factors Produced by the Analyzed Strains  
The strains obtained in pure culture were seeded on media to highlight soluble, enzymatic virulence factors. The expression of soluble enzymatic virulence factors was determined using eight tests that aimed to highlight the following enzymes: hemolysis, amylase, caseinase, gelatinase, production of esculinase, DNase, lipase, and lecithinase. _(a) Hemolysin production_  
The strains were seeded on agar with the addition of 5% sheep blood. After incubation at 37 \({}^{\circ}\)C for 24 h, the appearance of hemolysis areas around the microbial culture indicated the production of hemolysis (pore-forming toxins). _(b) Lipase production_  
The bacterial strains were seeded in _spot_ on agar with the addition of 1% Tween 80 (sorbitol 1-monooleate) and incubated for 72 h at 37 \({}^{\circ}\)C. The presence of an opaque precipitation zone around the culture spot, given by the formation of insoluble Ca oleate crystals (crystals formed between the released fatty acids and Ca\({}^{2+}\), was considered a positive reaction. _(c) Lecithinase production_  
The strains were seeded in spot on agar containing egg yolk (2.5%) and incubated for 72 h at 37 \({}^{\circ}\)C. The appearance of a clear area around the microbial culture was considered a positive reaction (lecithinase production). _(d) Protease production (caseinase and gelatinase)_  
The strains were seeded in spot on agar with the addition of 15% casein and 3% gelatin, respectively. After incubation at 37 \({}^{\circ}\)C, for 72 h, the production of proteases was indicated by the appearance of precipitation areas around the culture spots due to the formation of calcium paracasineate and transparent halos, respectively, due to the liquefaction of gelatin.