Basic concepts of bacterial identification, Microscopic morphological examination of bacteria

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1 Basic concepts of bacterial identification, Microscopic morphological examination of bacteria by: Zoltán Tigyi MD. Ph.D

2 CONTENT OF CHAPTERS TÉMA -The methods and steps of the identification of bacteria and microscopic morphological examination methods -The main light microscopic morphological methods -Aseptic / (sterile) technique -The schedule of practice -Preparation of bacterial smear from solid culture -Simple staining -Examination of the motility of bacteria -The theoretical background of Gram s staining -The Gram s staining procedure -Bacterial smears stained according different methods 2

3 METHODS AND STEPS OF IDENTIFICATION OF BACTERIA Identification of bacteria, is the process by which the bacteria species, and sometimes variants of intra-species (strain, isolate, clone, pathotype) is determined. The main reasons of the identification : 1. Clinical: - preparation for the application of appropriate therapy (e.g. administration of antitoxin in case of toxin producing bacteria) - the prediction of clinical course of the disease, especially in the case of specific infections causing bacteria (Neisseria meningitidis - meningitis, Streptococcus pneumoniae - pneumonia) - Blood-stream infection caused by Gram negative bacteriasepticaemia, endotoxin shock, etc. (see later) 3

4 METHODS AND STEPS OF IDENTIFICATION OF BACTERIA The main reasons of the identification : 2. Laboratory diagnostic : The bacterial species should be known for the proper evaluation of antibiotic susceptibility of bacteria, as this can be taken into account in the natural and acquired antibiotic resistance mechanisms. (see the practice on antibiotic susceptibility testing) 4

5 METHODS AND STEPS OF IDENTIFICATION OF BACTERIA 3. Public health, epidemiology and hospital hygiene and infection control: For choosing the most effective public health and infection control measures, e.g.: - isolation (quarantine) in case of highly infective pathogen e.g. (Corynebacterium diphtheriae, Neisseria meningitidis etc. ) - the usage of antimicrobial prophylaxis and prevention for persons at risk - Immunization: passive (gamma globulin) or active (vaccines) - reveal the chain of infection, reservoir, source of infection, vector, common vehicles (see: basic epidemiological concepts) 5

6 METHODS AND STEPS OF IDENTIFICATION OF BACTERIA PHENOTYPIC TESTING, (PHENOTYPING), Applications: - In the case of fast-growing bacteria, like the majority of human pathogens can be applied, as the method is based on the detection of enzymes involved in the cellular processes of life. - A new method is the mass spectrometry analysis of bacterial proteins profile. Which shortens the time of identification to some minutes, however, it is necessary to have pure culture. Overall, it can be one day shorter the time of identification. (MALDI-TOF MS Matrix-assisted laser desorption / ionization Time of Flight Mass Spectrometry) 6

7 METHODS AND STEPS OF IDENTIFICATION OF BACTERIA GENOTYPIC TESTING (GENOTYPING), Applications: - In case of non-culturable bacteria - on artificial media - (e.g. Chlamydophila spp.), hard to be grown, fastidious (e.g. some anaerobic bacteria) and slow-growing species (e.g. Mycobacterium tuberculosis etc.) - The method is based on the detection of the presence of species specific genes in the isolates or the DNA sequence analysis of the ribosomal genes. - The data gained by help of phenotype and genotype assays are compared to established databases thus the bacterial species can be specified. Especially, in the case of genotypic tests variants of intra-species (strain, isolate, clone, and 7 pathotype) also can be determined.

8 METHODS AND STEPS OF IDENTIFICATION OF BACTERIA THE TOOLS OF THE IDENTIFICATION - These are based on the individual characteristics of the microbes. Basic methods: 1, Microscopic morphological examination It is the examinations of native or stained preparations (smear of bacterial cell suspension on glass slide) by help of different types of light microscopes. The staining properties, the shape and the arrangement of the bacterial cells can be observed. (see details from slide no.: 23.) 8

9 METHODS AND STEPS OF IDENTIFICATION OF BACTERIA Basic methods: 2, Culturing, (cultivation): - By means of cultivation the bacterial growth potential can be examined under different nutritive and physical conditions (temperature, atmosphere etc.). - Examination of the bacterial growth potential on different basic media (e.g. nutrient agar, blood agar etc.) and diagnostic media i.e. selective and differential media (e.g. eosin methylene blue agar) (see later practice on culturing and bacterial media) and the observations of the colony morphology give important information for planning of further steps of the identification. 9

10 METHODS AND STEPS OF IDENTIFICATION OF BACTERIA Basic methods: 2, Culturing, (cultivation): - The major physical factors: - The optimum temperature is generally 37 O C, but can be higher e.g. Campylobacter spp. 42 O C, - Atmospheric conditions: aerobic (growth the presence of O 2 ) or anaerobic - (without O 2 ). - Duration of growth: 1-2 days in the case of fast-growing bacteria (e.g. Escherichia coli, Klebsiella pneumoniae, Streptococcus spp.), but the anaerobic bacteria 2-10 days. While the slow-growing bacteria need 4-8 weeks (e.g. 10 Mycobacterium tuberculosis etc.).

11 METHODS AND STEPS OF IDENTIFICATION OF BACTERIA Basic methods: 3, Biochemical tests: The metabolic characteristics of bacteria testing is performed on "Biochemical media" (BM), which are suitable to characterize the metabolic functioning of the bacterial cells. The existence or absence of a specific enzyme can be shown in a particular biochemical pathway by help of a certain BM. The pattern of results of biochemical tests is specific for a certain bacterial species as a fingerprint for an individual person. 11

12 METHODS AND STEPS OF IDENTIFICATION OF BACTERIA Basic methods: 3, Biochemical tests: By comparing the data gained from sufficient amounts of biochemical tests make possible to determine the species of the bacterium. This can be done manually or with semi or fully automated identifier machines. Details are on practice of "culturing and identification". Duration: By manually made, in the case of fast-growing bacteria, it takes 1-2 days, by automated identifier machine takes 4-6 hours. 12

13 METHODS AND STEPS OF IDENTIFICATION OF BACTERIA Basic methods: 4, Serological tests - It is the method when microbial surface antigens are detected by means of specific immune sera. - In some cases, an immediate diagnosis can be reached, at least up to the level of genus or sometimes to species. It is often used to supplement and corroborate the results of biochemical identification, e.g. determining serological types (serotypes), in case O, H, and K antigens. 13

14 THE METHODS AND STEPS OF THE IDENTIFICATION OF BACTERIA Basic methods: 4, Serological tests Duration: Depending on the method, e.g. glass-slide-, or latex bead agglutination tests takes 2-3 minutes, in this way, it serves the basis of many "fast", "quick" or " bedside" laboratory diagnostic tests. - However, e.g. agarose diffusion methods may last up to several days. - (Further details are on serologic practices and lectures.) 14

15 AUXILIARY METHODS OF BACTERIAL IDENTIFICATION It is often necessary to identify the isolates below species level (intra-species identification). For instance, it cannot be distinguished the non-pathogenic E. coli (commensal) form its pathogenic derivatives (E. coli pathotypes) by biochemical identification tests. In these cases auxiliary identification methods have to be used. (Further details are on lectures and practices.) 15

16 Auxiliary methods of bacterial identification 5, Examination of pathogenicity factors The pathogenicity factors are features that make the bacteria pathogenic to be able to cause disease in the host organism, such as exotoxins, exo-enzymes (e.g. coagulase of Staphylococcus aureus), adhesins (fimbria), haemagglutinins, etc.). Detection of type 3 fimbria of K. pneumoniae by bovine red blood cell Negative Positive 16

17 Auxiliary methods of bacterial identification 6, Bacteriophage typing - The epidemiological investigation requires intra-species identification. In this case the similarities or differences among the isolates from different samples should be demonstrated. - Bacteriophage infection is based on a specific receptorligand interaction. The different bacteriophage caused lytic patterns are compared of the isolates. If the phage lytic patterns are same of the different isolates then the common source is proved. 17

18 Auxiliary methods of bacterial identification 6, Bacteriophage typing of Staphylococcus aureus isolate Lytic zones on bacterial lawn. 18

19 Auxiliary methods of bacterial identification 7, Bacteriocin typing - Bacteriocins are proteins or peptide produced by bacteria and they kill the bacterial cells belong to the same or related species. - Their actions are based on specific receptor-ligand interaction. - Types of bacteriocins produced by the isolates and susceptibility patterns of the isolates against other bacteriocins make this method suitable for intra-species identification. - By comparison of different bacteriocin susceptibility patterns and the types of the produced bacteriocins of the different isolates give the opportunity to reveal whether the epidemiological relationships exist among them. 19

20 Auxiliary methods of bacterial identification 7, Bacteriocin typing Inhibitory zone of bacteriocin on bacterial lawn 20

21 Auxiliary methods of bacterial identification 8, Nucleic acid-based methods, geno-typing - Application fields: Non-culturable bacteria on artificial medium (e.g. Chlamydophila spp.), hard to be grown, fastidious species (e.g. certain anaerobic bacteria) or slowgrowing bacteria (e.g. Mycobacterium tuberculosis). - Below the level of species and species identification can be performed, even directly from clinical samples without having culture. 21

22 Auxiliary methods of bacterial identification 8, Nucleic acid-based methods, geno-typing - Since these methods generates huge amount of data, which made possible to establish a lot of different public open access databases e.g. PulsNet for Pulsed Field Gel Electrophoresis (PFGE), Multi-Locus Sequence Types database for MLST and gene banks for other DNA sequences. In this way, these methods are magnificently applicable in epidemiology and epidemiological studies to reveal the degree of relationship among isolates even world-wide. - Naturally, also possible to assess whether our local cases is an accidental cumulation or it is an epidemic. (Further details are on molecular biology practice.) 22

23 THE MAIN LIGHT MICROSCOPIC MORPHOLOGICAL METHODS LIGHT MICROSCOPIC NATIVE PREPARATION STAINED PREPARATION Light microscopy - Dark filed microscopy - Phase contrast, etc. - Living, unstained cells can be made visible. Simple staining: - Methylene blue - Fuchsine etc, -The shape and arrangement of the bacteria can be observed. Differential staining: - Gram - Ziehl-Neelsen or acid fast, Neisser etc. - Grouping the bacteria according to their staining features. Cell wall structure 23

24 MICROSCOPIC EXAMINATION OF NATIVE PREPARATIONS Native preparations: - They are used only in a narrow area of diagnostics since special microscopes are required and relatively little diagnostic information can be gained. Advantage: living bacterial cells can be observed. 24

25 EXAMINATION OF NATIVE PREPARATIONS Dark filed microscopy : - It requires special condenser and lighting from side direction, so only the scattering light entering object of the microscope. - The advantage of that is the high resolution 0,02 mm compared to the bright field microscope 0,2 mm. Thus the thin spirochete bacteria are hardly or not visible in brightfield microscope while it can be seen in dark-field microscope. In clinical samples the pathogens become visible such as Treponema pallidum of syphilis, Leptospira icterohaemorrhagiae of leptospirosis (field fever) Borrelia burgdorferi of Lyme-disease. (Further details are in Biophysics and Biology courses.) 25

26 EXAMINATION OF NATIVE PREPARATIONS Phase contrast microscope: - The advantage of that is a very contrasting picture of living cells, it is particularly useful for investigating very thin (0.1-1 mm), unstained preparations. - The disadvantage of that, especially in case of structures having high contras also generates a halo around them, which interferes with the imaging. - A special optical units called "phase shift ring" and above the objective so-called "condenser annulus" are required. The polarized property of the light wave and the differences between the optical density of the biological structures cause wave phase shift creating amplified image. (There are further details in biophysics and biology courses.) 26

27 EXAMINATION OF STAINED PREPARATIONS Simple staining: - The bacteria are stained by one type of dye e.g. methylene blue, or fuchsine etc. - The arrangement and shape of the stained bacterial cells can be examined with ordinary bright-field light microscopy. - Advantage: It is fast and cheap method. - Disadvantage: Information from bacterial cell wall type cannot be gained, however this knowledge is important for diagnostic and therapeutic reasons. 27

28 EXAMINATION OF STAINED PREPARATIONS Differential staining : - The bacteria are stained for at least two dyes, and a differentiating solution is used which more effectively solves the bound dye molecules from certain cell wall components than the other components. The second dye stains the decolorized cell wall components. - Advantage: Beyond the shape and arrangement of the bacterial cells the cell wall type also can be determined. - Disadvantage: Some of dyes and solutions are toxic e.g. Gram-dyes. 28

29 ASEPTIC / (STERILE) TECHNIQUE - All manipulation should be performed within the 30 cm circle around the flame of the Bunsen-burner. The rising air will help to prevent the entrance of dust particles upon which contaminating bacteria may present. - Every time when you use the loop, it has to flam it to be sterile. Heat the loop until it glows then let it cool down (~20 seconds) before touching the culture. - Flaming the mouth of test tube after unplugging and before plugging. - While the tubes are open, hold them at a 45 degree angle so that dust cannot fall into the open tube. - The purpose of flaming is not to sterilize, but to warm the tube and create warm air convection currents up and away from the 29 opening.

30 ASEPTIC / (STERILE) TECHNIQUE - The test-tube is held in the left hand (for a right-handed person). The instrument (loop, pipet, or needle) is held in the right hand. - The test-tube cap is grasped by the little- and ring-finger of the right hand, and removed. - Do not put the plug of the test tube on the table because contamination may occur. (In both directions, from the culture to environment and vice versa.) - While continuing to hold the cap with the fingers, the tube is lightly flamed and the instrument is manipulated appropriately. - The cap is replaced on the test-tube and the test-tube is put back into the rack. 30

31 ASEPTIC / (STERILE) TECHNIQUE - Petri dish lids prevent dust from falling directly onto plates but allow diffusion of air around the edges. - Do not place the lid on the bench top. Do not leave plates uncovered. - Do not walk around the room with an open plate. 31

32 ASEPTIC / (STERILE) TECHNIQUE All manipulations should be performed within the 30 cm circle around the flame of the Bunsen-burner. Every time when you use the loop, it has to flam it to be sterile. Heat the loop until it glows then let it cool down (~20 seconds) before touching the culture. 32

33 ASEPTIC / (STERILE) TECHNIQUE The test-tube cap is grasped by the little- and ring-finger of the right hand, and removed. - Flaming the mouth of test tube after unplugging and before plugging. 33

34 ASEPTIC / (STERILE) TECHNIQUE - While the tubes are open, hold them at a 45 degree angle so that dust cannot fall into the open tube. - Do not put the plug of the test tube on the table. 34

35 MICROSCOPIC MORPHOLOGICAL EXAMINATION TASKS: 1 st DAY I. Simple staining. 2 nd. DAY: II. Examination of the motility of bacteria on native preparation. III. Differential or combined staining, Gram-staining. 35

36 PREPARATION OF BACTERIAL SMEAR 1.Degrease the glass slide in gas flame, passing it over the flame 3 times. 36

37 PREPARATION OF BACTERIAL SMEAR 2. Put one drop of saline on the middle of the glass-slide. 37

38 PREPARATION OF BACTERIAL SMEAR 3. Preparation of bacterial cell suspension. With sterilised and cooled loop pick up some colonies then transfer a minute quantity of bacteria beside the physiological saline solution. 38

39 PREPARATION OF BACTERIAL SMEAR 3. Preparation of bacterial cell suspension. Gradually mix them together with circular movement of the loop till getting even suspension. 39

40 PREPARATION OF BACTERIAL SMEAR 4. Allow the smear to air dry. Do not heat it directly in the flame. However, it is faster the drying by holding it into the rising hot air stream above the flame approximately at height of cm. 40

41 PREPARATION OF BACTERIAL SMEAR 5. Fix the bacterial cell onto the surface of the glass-slide by slowly passing the completely dried preparation 3 times over the flame. 41

42 MICROSCOPIC MORPHOLOGICAL EXAMINATION SIMPLE STAINING OF BACTERIA Materials and devices: - Nutrient agar culture of Escherichia coli; Bacillus cereus; Staphylococcus aureus, and Candida albicans - physiological saline solution; - glass slide; - staining rack; - Gram's solutions; - inoculating loop; - Bunsen-burner; - light microscope, - immersion oil, - blotting-paper. 42

43 MICROSCOPIC MORPHOLOGICAL EXAMINATION PROCESSING OF SIMPLE STAINING : - Cover the heat fixed bacterial smear with 3-5 drops of either methylene blue or fuchsine solution. 43

44 MICROSCOPIC MORPHOLOGICAL EXAMINATION PROCESSING OF SIMPLE STAINING : - Incubate the smear with dye solution for 3 minutes. - Then wash it with water carefully. 44

45 MICROSCOPIC MORPHOLOGICAL EXAMINATION PROCESSING OF SIMPLE STAINING : - Pour off the excess water from the smear then place it onto the strip of the blotting-paper on the bench, then cover it with the other half of the blotting-paper and blot the smear carefully to avoid of removing the bacterial cell from the smear. 45

46 MICROSCOPIC MORPHOLOGICAL EXAMINATION PROCESSING OF SIMPLE STAINING : - Do not wipe it because the bacteria may be removed from the smear. - Allow it to dry. 46

47 MICROSCOPIC MORPHOLOGICAL EXAMINATION MICROSCOPY : - The smear is examined with using of 100 x magnification immersion objective. 47

48 MICROSCOPIC MORPHOLOGICAL EXAMINATION MICROSCOPY: - Drop one drop of immersion oil onto the smear and examine with 100 x oil immersion objective lens. 48

49 MICROSCOPIC MORPHOLOGICAL EXAMINATION MICROSCOPY : - Decrease the working distance between the objective lens and glass-slide carefully immersing the objective in the oil but just not to touch the glass-slide. - After that, increase the working distance between the objective lens and glass-slide carefully by slowly screwing the micrometer screw in the opposite direction while looking for sharp image. 49

50 MICROSCOPIC MORPHOLOGICAL EXAMINATION MICROSCOPY : - After using the microscope wipe the immersion lens with a piece of cloth previously moistened with 1-2 drop of xylene (xylol). Place back the dust cover onto the microscope. 50

51 MICROSCOPIC MORPHOLOGICAL EXAMINATION TASKS 2 nd DAY: II. Examination of the motility of bacteria on native preparation. III. Differential or combined staining, Gram-staining. 51

52 MICROSCOPIC MORPHOLOGICAL EXAMINATION, NATIVE PREPARATION - The ciliated bacteria can exhibit motility i.e. self-propelled motion, under appropriate circumstances, which is guided by either positive stimuli e.g. nutrients and then bacterial cell approach that or negative stimuli e.g. toxic compounds and then bacterial cell back away. Bacterial cells are able to detect the concentrations of such compounds that determine the direction of the bacterial migration. - Active movement of the bacterial cell has a definite direction, while Brownian motion is more similar to resonance and no definite direction. So called Brownian motion is due to random molecular bombardment of tiny bacterial cells by the molecules of the solvent. - Many times the un-even drying out the preparation may casus the movement of the liquid film, then all cells flowing 52 in one direction at a time.

53 MICROSCOPIC MORPHOLOGICAL EXAMINATION, NATIVE PREPARATION - Some ciliated bacteria capable for swarming i.e. bacterial cell migrate onto the surface of the solid medium. For expressing of this attribute bacterial cell needs further genes than beyond the gens of cilia, in this way, not all the ciliated bacteria are able to swarm. - Notice the swarming of Proteus on the surface of the nutrient agar plate. 53

54 EXAMINATION OF BACTERIAL MOTILITY 1. Apply the sterile technique! (See slides ) 3-5 loop-full of liquid cultures should be placed in the middle of the slide. Only the closed inoculation loop is suitable for picking up liquid film. Do not forget flaming the inoculation loop at every turn of placing the culture on the glass-slide. 54

55 EXAMINATION OF BACTERIAL MOTILITY 2. The covering of culture drops by cover slip: Touch the drop with the edge of the cover-slip and then allow running the fluid along the edge. After this, gradually approaches that side of the cover-slip where we grabbed, and when the distance between the cover-slip and that glass slide is approx. 2-3 mm, then "drop" the cover-slip onto the drop of culture on the glass slide. When it was done properly, you will have no or minimal bubble under the cover-slip. 55

56 EXAMINATION OF BACTERIAL MOTILITY 3. The preparation should be examined with 40 x objective lens, with narrowed diaphragm. - Look for actively moving bacteria. 3-4 per field of vision actively moving bacteria can be expected. The majority of Brownian motion carried. 56

57 EXAMINATION OF BACTERIAL MOTILITY 57

58 Differentiating or complex staining Gram s staining procedure Hans Christian Joachim Gram, ( ) Danish bacteriologist, - Staining procedure published in 1884 in Berlin -Source: - - His staining method made it possible to classify the bacteria. 58

59 GRAM S STAINING PROCEDURE I. PRIMARY STAINING : Stain with gentian-violets (crystal violet) cells are stained violet colour. II. COMPLEX FORMATION: GRAM'S IODINE = lugol solution -It serves as a mordant process (curing). It forms an insoluble complex by binding to the primary stain and this complex associate with the peptidoglycan layers of the cell wall. The iodine-dye-peptidoglycan complex formation occurs in the cell wall of the bacterial cell wall. Gram (+) peptidoglycan layers Gram (-) 1-2 peptidoglycan layers 59

60 GRAM S STAINING PROCEDURE 3. DECOLOURIZATION, DIFFERENTIATION: acetone-alcohol - dissolves the crystal violet from the peptidoglycan, - during the same time, same quantity of dye comes out Gram (+) Retains most of the gentianviolet, it remains violet peptidoglycan layers Gram (-) It lost the dye. It is colourless. 1-2 layers only. 4. COUNTER STAIN: fuchsine or safranin It stains red those cells that have been previously decolourized. Gram (+) violet Gram (-) red 60

61 Schematic structure of Gram negative cell wall Porin channel Protein bound to cytoplasmic membrane Lipopolysaccharide,LPS Outer membrane Peptidoglycan layers Periplasmic space Cytoplasmic membrane 61

62 Schematic structure of Gram positive cell wall Proteins associated with the cell wall Lipoteichoic acid Teichoic acid Peptidoglycan layers Cytoplasmic membrane 62

63 Differentiating or complex staining, Gram s staining procedure Gram's solutions 63

64 Differentiating or complex staining, Gram s staining procedure 1. PRIMARY STAINING: The smear it was prepared according to the process of the preparation of bacterial smear, is covered with crystal violet solution. Incubation: 1 minute 64

65 Differentiating complex or staining, Gram s staining procedure 2. RINSE: with water. 65

66 Differentiating or complex staining, Gram s staining procedure 3. COMPLEX FORMATION : covers with Gram s iodine solution Incubation: 2 minutes 66

67 Differentiating or complex staining, Gram s staining procedure 4. RINSE: with water 67

68 Differentiating or complex staining, Gram s staining procedure 5. DIFFERENTIATION OR DECOLOURIZATION WITH ACETONE- ALCOHOL: This is the most crucial step. Hold the smear at an angle and add acetone-alcohol dropwise to the stained bacteria covered area until the crystal violet intensively dissolves. Generally 5-10 seconds, 5-10 drops enough depending of the bacterial cell density. DO NOT OVER-DECOLOURIZE IT MAY RESULT FALSE RESULT. 68

69 Differentiating or complex staining, Gram s staining procedure 6. STOPPING THE REACTION AND RINSING: with water. 69

70 Differentiating complex or staining, Gram s staining procedure 7. COUNTER STAINING: covers with fuchsine solution 70

71 Differentiating or complex staining, Gram s staining procedure 8. RINSE: with water 71

72 Differentiating or complex staining, Gram s staining procedure - 9. Drying: Pour off the excess water from the smear then place it onto the strip of the blotting-paper on the bench, then cover it with the other half of the blotting-paper and blot the smear carefully. Do not wipe it because the bacteria may be removed from the smear. - Allow it to dry. 72

73 Differentiating or complex staining, Gram s staining procedure - MICROSCOPY: - Drop one drop of immersion oil onto the smear and examine with 100 x oil immersion objective lens. 73

74 BACTERIAL SMEARS STAINED ACCORDING TO GRAM Staphylococcus aureus, Gram (+) coccus Haemophilus influenzae, Gram (-) coccus 74

75 BACTERIAL SMEARS STAINED ACCORDING TO GRAM Bacillus cereus, Gram (+) rod Escherichia coli, Gram (-) rod 75

76 BACTERIAL SMEARS STAINED ACCORDING TO GRAM Bacillus cereus, Gram (+) rods from 48 hours culture. The cells were not stained positively according to Gram they were in the advanced phases of endospore formation. Non staining part is the endospore. 76

77 BACTERIAL SMEARS STAINED ACCORDING TO GRAM Klebsiella pneumoniae, Gram (-) rod 77

78 BACTERIAL SMEARS STAINED ACCORDING TO GRAM Candida albicans, a budding fungal cell, yeast like fungus which stains Gram positively. The fungal cell wall also contains compounds that form complex with crystal violet but it does not have true bacterial Gram (+) cell wall structure. 78

79 BACTERIAL SMEARS STAINED ACCORDING TO GRAM Gram (+) cocci can be seen among the red blood cells. Preparation from blood culture (haemoculture). 79

80 BACTERIAL SMEARS STAINED ACCORDING TO GRAM Alveolar sample. Gram (-) rods. Non-stained (negatively stained) area around the rods is the microscopic sign of the capsule formation. Which not stainable according to Gram. Klebsiella pneumoniae grew from the sample which is an encapsulated bacteria. Alveolar macrophage is phagocytizing a bacterial cell. Non-stained (negatively stained) area around the rods is the microscopic sign of the capsule formation. 80

81 Ziehl-Neelsen or acid fast staining Mycobacterium tuberculosis bacteria in a sputum from a patient having open tuberculosis. Small red rods in clusteres in bluish background, characteristic appearance of Ziehl- Neelsen staining. This staining procedure is not species specific therefore the diagnosis can be acid fast (rod) bacterium positive. 81

82 Neisser s staining Corynebacterium diphtheriae, was stained according to Neisser. This procedure stains polyphosphate granules in metachromatic manner, it is called as polar granules or Babes- Ernst granules. It can be seen as a reddish dots one end of the small rods. This is not a specific staining procedure for Corynebacterium diphtheriae. 82

83 ACKNOWLEDGEMENTS - For Dr. Gábor Smuk, (University of Pécs, Clinical Centre, Department of Pathology), who made possible and helped me to take microscopic pictures (micro photos) from the smear in high quality and resolution and shoot video from actively moving bacterial cells. - For Horváth Marianna, (UP. CC. Department of Medical Microbiology and Immunology), Ph.D. student, who helped me to take a picture of all the other photos. - For Levente Emődy, (UP. CC. Department of Medical Microbiology and Immunology), who proofread the translation. 83