Culture

Material and Methods

Material

Cancer patients with prickle cell carcinoma of esophagus, malignant melanoma, breast cancer (4 cases), malignant lymphogranuloma ( 2 cases), gastric carcinoma, bronchial carcinoid, hepatoma, liver cancer, malignant lymphoma, bladder cancer, sarcoma, renal cell carcinoma, colon carcinoma, basalioma (2 cases), carcinoma of the tongue, carcinoma of the prostate, rectal carcinoma, esophageal cancer, leukemia, melanoma. Control patients with hepatitis, hyperuricemia, pyelitis, lues, hypertension, psoriasis, chr. tonsillitis, nephrolittiasis and 5 healthy persons. For the diagnosis and the material I have to thank R. Pekar.

Methods

Culture medium

  1. 35g yeast incubated in 25ml 20% KOH-solution for 24 h at 36°C
  2. 10ml of the suspension diluted in 50ml aqua  dest.
  3. 6g bacto beef (Difco) in 12ml 20% KOH-solution
  4. 0,7g proteose pepton (Difco) in 20ml aqua dest.
  5. Incubation of the mixture of the solutions 3 and 4  for 6h at 36°C
  6. 1g D(+)-glucose (Merck) in 10ml aqua dest.
  7. 5ml of a mixture of the solutions 2,5,6 in sterilized culture tubes
  8. pH-value of the culture medium is 12,5

Culture

  1. 1ml heparin anticoagulated peripheral blood was suspended in 2ml 7,5% KOH-solution and incubated for 8h at 37°C
  2. 0,5ml of the sediment was put in the culture tubes with the culture medium
  3. incubution under aerobic conditions at 30°C for 7 weeks

Preparation

  1. Centrifugation of the fluid of the cultures at 3500U/ 15min
  2. fixation of the fluid in Carnoy’s fixative
  3. staining of the smear of the fluid with Giemsa
  4. preparations for the SEM and TEM

Further culture

  1. Mycoplasma-agar (Merck) with inactivated Mycoplasma-free lamb serum
  2. Mycoplasma bouillon (Merck) with inactivated Mycoplasma-free lamb serum

Results

In the group of cancer patients we had 90% positive, 6% insecure, 4% negative cultures (n=48). In the group of patients with other diseases we had 25% positive and 75% negative cultures. In the group of healthy persons we found 20% positive and 80% negative cultures. We used for the analyses Giemsa stained smears and a magnification of 1600 : 1 (fig. 4 and fig. 5).

On agar plates we found two culture types, rough cultures and fried egg cultures (fig. 6a and fig. 6b).

In bouillon cultivated particles can be seen with dark field microscopy (fig.7). With SEM we found 0,25µm particles and ring forms (fig. 8 and fig. 9). With the TEM we found 0,25µm cell-wall-deficient particles (fig. 10) ,0,25µm particles with a cell wall (fig.10a) and cross sections through ring forms (fig. 11).

 

Fig.4 Particles (Basoplasma sanguineum), breast cancer (44a), Giemsa staining, magnification 1600 : 1, positive. (higher resolution)

 

Fig.5 Particles (Basoplasma sanguineum),prickle cell carcinoma of esophagus, Giemsa staining, magnification 1600 : 1, positive. (higher resolution)

 

Fig.6a Colonies of the particles (Basoplasma sanguineum) on Mycoplasma – agar (Merck) with Mycoplasma-free inactivated lamb serum. (higher resolution)

 

Fig.6b Colonies of the particles (Basoplasma sanguineum) on Mycoplasma – agar (Merck) with Mycoplasma-free inactivated lamb serum.

 

Fig.7 Culture of the particles (Basoplasma sanguineum) in Mycoplasma – bouillon (Merck) with Mycoplasma-free inactivated lamb serum, breast cancer, darkfield, magnification 1600 : 1. ( higher resolution)

 

Fig.8 Particles (Basoplasma sanguineum) of a positive culture (prickle cell carcinoma of esophagus) ,MF- method, REM, magnification 6250 : 1. (higher resolution)

 

Fig.9 Particles (Basoplasma sanguineum) of a positive culture (breast cancer) directly smeared on a cover plate, REM, magnification 6250 : 1 . (higher resolution)

Fig.10 Particles (Basoplasma sanguineum), cell- wall-deficient, of a positive culture (prickle cell carcinoma of esophagus), TEM, magnification 25 000:1. (higher resolution)

 

Fig.10a Particles (Basoplasma sanguineum) with cell wall, of a positive culture (prickle cell carcinoma of esophagus), TEM, magnification 25 000:1. (higher resolution)

 

Fig. 11 Particles (Basoplasma sanguineum) of a positive culture (breast cancer), TEM, magnification 25 000:1. (higher resolution)

 

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